1-pyridazin-/triazin-3-yl-piper(-azine)/idine/pyrolidine derivatives and compositions thereof for inhibiting the activity of shp2

ABSTRACT

The present invention relates to compounds of formula I: 
     
       
         
         
             
             
         
       
     
     in which m, Y 1 , Y 2 , Y 3 , R 1 , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5a  and R 5b  are defined in the Summary of the Invention; capable of inhibiting the activity of SHP2. The invention further provides a process for the preparation of compounds of the invention, pharmaceutical preparations comprising such compounds and methods of using such compounds and compositions in the management of diseases or disorders associated with the aberrant activity of SHP2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/117,674 filed 30 Aug. 2018, which is a continuation of U.S. patentapplication Ser. No. 15/110,498, filed 8 Jul. 2016, which is a 371 U.S.national phase application of international application numberPCT/IB2015/050343 filed 16 Jan. 2015, which application claims thebenefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication No. 61/928,738, filed 17 Jan. 2014. The disclosure of theseapplications are incorporated herein by reference in their entirety andfor all purposes.

BACKGROUND Field of the Invention

The present invention relates to compounds capable of inhibiting theactivity of SHP2. The invention further provides a process for thepreparation of compounds of the invention, pharmaceutical preparationscomprising such compounds and methods of using such compounds andcompositions in the management of diseases or disorders associated withthe aberrant activity of SHP2.

Background of the Invention

The Src Homolgy-2 phosphatase (SHP2) is a non-receptor protein tyrosinephosphatase encoded by the PTPN11 gene that contributes to multiplecellular functions including proliferation, differentiation, cell cyclemaintenance and migration. SHP2 is involved in signaling through theRas-mitogen-activated protein kinase, the JAK-STAT or thephosphoinositol 3-kinase-AKT pathways.

SHP2 has two N-terminal Src homology 2 domains (N-SH2 and C-SH2), acatalytic domain (PTP), and a C-terminal tail. The two SH2 domainscontrol the subcellular localization and functional regulation of SHP2.The molecule exists in an inactive, self-inhibited conformationstabilized by a binding network involving residues from both the N-SH2and PTP domains. Stimulation by, for example, cytokines or growthfactors leads to exposure of the catalytic site resulting in enzymaticactivation of SHP2.

Mutations in the PTPN11 gene and subsequently in SHP2 have beenidentified in several human diseases, such as Noonan Syndrome, LeopardSyndrome, juvenile myelomonocytic leukemias, neuroblastoma, melanoma,acute myeloid leukemia and cancers of the breast, lung and colon. SHP2,therefore, represents a highly attractive target for the development ofnovel therapies for the treatment of various diseases. The compounds ofthe present invention fulfill the need of small molecules to thatinhibit the activity of SHP2.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds of Formula I:

in which: m is selected from 0, 1 and 2; p is selected from 0 and 1; Y₁is selected from CH and N; Y₂ is selected from CR₆ and N; Y₃ is selectedfrom NH and CR₇R₈; R₁ is selected from C₆₋₁₀aryl, C₃₋₈cycloalkyl,C₃₋₈cycloalkenyl and a 5-9 member heteroaryl group containing from 1 to4 heteroatoms selected from N, O and S; wherein said aryl or heteroarylof R₁ is substituted with 1 to 5 R₉ groups independently selected fromhalo, amino, hydroxy, N₃, C₁₋₄alkyl, hydroxy-substituted-C₁₋₄alkyl,halo-susbtituted-C₁₋₄alkyl, amino-substituted-C₁₋₄alkyl, —C(O)OR₁₀ and—NHC(O)R₁₀; R₁₀ is selected from hydrogen, phenyl and naphthyl; whereinsaid phenyl of R₁₀ is unsubstituted or substituted with methoxy; R_(2a)and R_(2b) are independently selected from hydrogen, C₁₋₄alkyl,C₁₋₄alkoxy, amino, hydroxy, C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R_(3a)and R_(3b) are independently selected from hydrogen, halo, carbonyl,C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy, C₃₋₈cycloalkyl andC₁₋₄alkyl-amino; R_(4a) and R_(4b) are independently selected fromhydrogen, halo, carbonyl, C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy,C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R_(5a) and R_(5b) are independentlyselected from hydrogen, carbonyl, C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy,C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; wherein any two groups selected fromR_(2a), R_(3a), R_(4a) and R₇ can form a 5 to 6 member unsaturated,partially unsaturated ring or saturated ring optionally containing aring nitrogen; R₆ is selected from hydrogen, halo, cyano, C₁₋₄alkylC₁₋₄alkoxy, amino-carbonyl, halo-substituted C₁₋₄alkyl, halo-substitutedC₁₋₄alkoxy, hydroxy-substituted C₁₋₄alkyl, amino-substituted C₁₋₄alkyl,—S(O)₁₋₂R_(6a), —C(S)R_(6a), —C(O)NR_(6a)R_(6b), —C(NH)NR_(6a)R_(6b) and—NR_(6a)C(O)R_(6b); wherein R_(6a) and R_(6b) are independently selectedfrom hydrogen and C₁₋₄alkyl; R7 is selected from hydrogen, C₁₋₄alkyl,C₃₋₆cycloalkyl, C₆₋₁₀aryl and a 5-9 member heteroaryl group containingfrom 1 to 4 heteroatoms selected from N, O and S; R₇ and R_(4a) togetherwith the carbon atoms to which they are both attached can form a fusedpyrrolidinyl or cyclopropyl group substituted with amino (such asexample 19, infra); R₈ is selected from amino, amino-methyl andmethyl-amino; or a pharmaceutically acceptable salt thereof.

In a second aspect, the present invention provides a pharmaceuticalcomposition which contains a compound of Formula I or a N-oxidederivative, tautomer, individual isomers and mixture of isomers thereof;or a pharmaceutically acceptable salt thereof, in admixture with one ormore suitable excipients.

In a third aspect, the present invention provides a method of treating adisease in an animal in which modulation of SHP2 activity can prevent,inhibit or ameliorate the pathology and/or symptomology of the diseases,which method comprises administering to the animal a therapeuticallyeffective amount of a compound of Formula I or a N-oxide derivative,individual isomers and mixture of isomers thereof, or a pharmaceuticallyacceptable salt thereof.

In a fourth aspect, the present invention provides a method of treatinga disease in an animal in which modulation of SHP2 activity can prevent,inhibit or ameliorate the pathology and/or symptomology of the diseases,which method comprises administering to the animal a therapeuticallyeffective amount of a compound of Formula I or a N-oxide derivative,individual isomers and mixture of isomers thereof, or a pharmaceuticallyacceptable salt thereof, in simultaneous or sequential combination withan anti-cancer therapeutic.

In a fifth aspect, the present invention provides the use of a compoundof Formula I in the manufacture of a medicament for treating a diseasein an animal in which SHP2 activity contributes to the pathology and/orsymptomology of the disease.

In a sixth aspect, the present invention provides a process forpreparing compounds of Formula I and the N-oxide derivatives, prodrugderivatives, protected derivatives, individual isomers and mixture ofisomers thereof, and the pharmaceutically acceptable salts thereof.

Definitions

The general terms used hereinbefore and hereinafter preferably havewithin the context of this disclosure the following meanings, unlessotherwise indicated, where more general terms whereever used may,independently of each other, be replaced by more specific definitions orremain, thus defining more detailed embodiments of the invention:

“Alkyl” refers to a fully saturated branched or unbranched hydrocarbonmoiety having up to 20 carbon atoms. Unless otherwise provided, alkylrefers to hydrocarbon moieties having 1 to 7 carbon atoms (C₁₋₇alkyl),or 1 to 4 carbon atoms (C₁₋₄alkyl). Representative examples of alkylinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like. Asubstituted alkyl is an alkyl group containing one or more, such as one,two or three substituents selected from halogen, hydroxy or alkoxygroups. Halo-substituted-alkyl and halo-substituted-alkoxy, can beeither straight-chained or branched and includes, methoxy, ethoxy,difluoromethyl, trifluoromethyl, pentafluoroethyl, difluoromethoxy,trifluoromethoxy, and the like.

“Aryl” means a monocyclic or fused bicyclic aromatic ring assemblycontaining six to ten ring carbon atoms. For example, aryl may be phenylor naphthyl, preferably phenyl. “Arylene” means a divalent radicalderived from an aryl group.

“Heteroaryl” is as defined for aryl above where one or more of the ringmembers is a heteroatom. For example C₅₋₁₀heteroaryl is a minimum of 5members as indicated by the carbon atoms but that these carbon atoms canbe replaced by a heteroatom. Consequently, C₅₋₁₀heteroaryl includespyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl,benzopyranyl, benzothiopyranyl, benzo[1,3]dioxole, imidazolyl,benzo-imidazolyl, pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl,tetrazolyl, pyrazolyl, thienyl, etc.

“Cycloalkyl” means a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing the numberof ring atoms indicated. For example, C₃₋₁₀cycloalkyl includescyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, etc.

“Heterocycloalkyl” means cycloalkyl, as defined in this application,provided that one or more of the ring carbons indicated, are replaced bya moiety selected from —O—, —N═, —NR—, —C(O)—, —S—, —S(O)— or —S(O)₂—,wherein R is hydrogen, C₁₋₄alkyl or a nitrogen protecting group. Forexample, C₃₋₈heterocycloalkyl as used in this application to describecompounds of the invention includes morpholino, pyrrolidinyl,pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone,1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, thiomorpholino, sulfanomorpholino,sulfonomorpholino, etc.

“Halogen” (or halo) preferably represents chloro or fluoro, but may alsobe bromo or iodo.

“SHP2” means “Src Homolgy-2 phosphatase” and is also known as SH-PTP2,SH-PTP3, Syp, PTP1D, PTP2C, SAP-2 or PTPN11.

Cancers harboring “PTPN11 mutations” include but are not limited to:N58Y; D61Y, V; E69K; A72V, T, D; E76G, Q, K (ALL); G60A; D61Y; E69V;F71K; A72V; T73I; E76G, K; R289G; G503V (AML); G60R, D61Y, V, N; Y62D;E69K; A72T, V; T731; E76K, V, G, A, Q; E139D; G503A, R; Q506P (JMML);G60V; D61V; E69K; F71L; A72V; E76A (MDS); Y63C (CMML); Y62C; E69K; T507K(neuroblastoma); V46L; N58S; E76V (Lung cancer); R138Q (melanoma); E76G(colon cancer).

Compounds of formula I may have different isomeric forms. For example,any asymmetric carbon atom may be present in the (R)-, (S)- or(R,S)-configuration, preferably in the (R)- or (S)-configuration.Substituents at a double bond or especially a ring may be present incis- (=Z-) or trans (=E-) form. The compounds may thus be present asmixtures of isomers or preferably as pure isomers, preferably as purediastereomers or pure enantiomers.

Where the plural form (e.g. compounds, salts) is used, this includes thesingular (e.g. a single compound, a single salt). “A compound” does notexclude that (e.g. in a pharmaceutical formulation) more than onecompound of the formula I (or a salt thereof) is present, the “a” merelyrepresenting the indefinite article. “A” can thus preferably be read as“one or more”, less preferably alternatively as “one”.

Wherever a compound or compounds of the formula I are mentioned, this isfurther also intended to include N-oxides of such compounds and/ortautomers thereof.

The term “and/or an N-oxide thereof, a tautomer thereof and/or a(preferably pharmaceutically acceptable) salt thereof” especially meansthat a compound of the formula I may be present as such or in mixturewith its N-oxide, as tautomer (e.g. due to keto-enol, lactam-lactim,amide-imidic acid or enamine-imine tautomerism) or in (e.g. equivalencyreaction caused) mixture with its tautomer, or as a salt of the compoundof the formula I and/or any of these forms or mixtures of two or more ofsuch forms.

For the following compounds, the NH2 group attached to the pyrazine ringis critical for potency. Analysis of the crystallographic structureshows the NH2 group in an intramolecular interaction with the backbonecarbonyl group of SHP2 residue E250:

SHP2 Compound IC50

70 nM

 5.7 μM

The present invention also includes all suitable isotopic variations ofthe compounds of the invention, or pharmaceutically acceptable saltsthereof. An isotopic variation of a compound of the invention or apharmaceutically acceptable salt thereof is defined as one in which atleast one atom is replaced by an atom having the same atomic number butan atomic mass different from the atomic mass usually found in nature.Examples of isotopes that may be incorporated into the compounds of theinvention and pharmaceutically acceptable salts thereof include, but arenot limited to, isotopes of hydrogen, carbon, nitrogen and oxygen suchas as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³⁵S, ¹⁸F, ³⁶Cl and ¹²³I.Certain isotopic variations of the compounds of the invention andpharmaceutically acceptable salts thereof, for example, those in which aradioactive isotope such as ³H or ¹⁴C is incorporated, are useful indrug and/or substrate tissue distribution studies. In particularexamples, ³H and ¹⁴C isotopes may be used for their ease of preparationand detectability. In other examples, substitution with isotopes such as²H may afford certain therapeutic advantages resulting from greatermetabolic stability, such as increased in vivo half-life or reduceddosage requirements. Isotopic variations of the compounds of theinvention or pharmaceutically acceptable salts thereof can generally beprepared by conventional procedures using appropriate isotopicvariations of suitable reagents. For example, compound can exist in adeutorated form as shown below:

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to compounds capable of inhibiting theactivity of SHP2. In one aspect of the invention, with respect tocompounds of Formula I, are compounds of Formula Ia:

in which: m is selected from 0 and 1; n is selected from 1, 2, 3, 4 and5; Y₁ is selected from CH and N; Y₂ is selected from CR₆ and N; R_(4a)is selected from hydrogen, methyl and hydroxy; R₆ is selected fromhydrogen, halo, cyano, C₁₋₄alkyl, C₁₋₄alkoxy, amino-carbonyl,halo-substituted C₁₋₄alkyl, halo-substituted C₁₋₄alkoxy,hydroxy-substituted C₁₋₄alkyl, amino-substituted C₁₋₄alkyl,—S(O)_(1-2a)R_(6a), —C(S)R_(6a), —C(O)NR_(6a)R_(6b), —C(NH)NR_(6a)R_(6b)and —NR_(6a)C(O)R_(6b); wherein R_(6a) and R_(6b) are independentlyselected from hydrogen and C₁₋₄alkyl; R₇ is selected from hydrogen,methyl, phenyl, pyrazinyl and pyridinyl; R₇ and R_(4a) together with thecarbon atoms to which they are both attached can form a cyclopropylgroup substituted with amino; R₈ is selected from amino andmethyl-amino; R₉ is selected from halo, amino, hydroxy, N₃, C₁₋₄alkyl,halo-substituted-C₁₋₄alkyl, C₁₋₄alkoxy, —C(O)OR₁₀ and —NHC(O)R₁₀; R₁₀ isselected from hydrogen, phenyl and naphthyl; wherein said phenyl of R₁₀is unsubstituted or substituted with methoxy; or a pharmaceuticallyacceptable salt thereof.

In a further aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

In another aspect of the invention are compounds of formula Ib:

in which: Y₁ is selected from CH and N; Y₂ is selected from CR₆ and N;R₁ is selected from thiophen-2-yl and 1H-indol-7-yl; wherein saidthiophen-2-yl can be substituted with a group selected from methyl andchloro; R₆ is selected from hydrogen, halo and methyl; R₈ is selectedfrom amino and methyl-amino; or a pharmaceutically acceptable saltthereof.

In a further aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

In another aspect of the invention are compounds of formula Ic:

in which: n is selected from 1, 2, 3, 4 and 5; Y₁ is selected from CHand N; Y₂ is selected from CR₆ and N; R_(3a) is selected from C₁₋₄alkyl,C₁₋₄alkoxy, amino, hydroxy, C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R_(4a)is selected from hydrogen, C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy,C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R₆ is selected from hydrogen, halo,cyano, C₁₋₄alkyl, C₁₋₄alkoxy, amino-carbonyl, halo-substitutedC₁₋₄alkyl, halo-substituted C₁₋₄alkoxy, hydroxy-substituted C₁₋₄alkyl,amino-substituted C₁₋₄alkyl, —S(O)₁₋₂R_(6a), —C(S)R_(6a),—C(O)NR_(6a)R_(6b), —C(NH)NR_(6a)R_(6b) and —NR_(6a)C(O)R_(6b); whereinR_(6a) and R_(6b) are independently selected from hydrogen andC₁₋₄alkyl; R₁₀ is selected from hydrogen, phenyl and naphthyl; whereinsaid phenyl of R₁₃ is unsubstituted or substituted with methoxy; or apharmaceutically acceptable salt thereof.

In a further aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

In another aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

In another aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

Pharmacology and Utility

The Src Homolgy-2 phosphatase (SHP2) is a protein tyrosine phosphataseencoded by the PTPN11 gene that contributes to multiple cellularfunctions including proliferation, differentiation, cell cyclemaintenance and migration. SHP2 is involved in signaling through theRas-mitogen-activated protein kinase, the JAK-STAT or thephosphoinositol 3-kinase-AKT pathways. SHP2 mediates activation of Erkland Erk2 (Erkl/2, Erk) MAP kinases by receptor tyrosine kinases such asErbBl, ErbB2 and c-Met.

SHP2 has two N-terminal Src homology 2 domains (N-SH2 and C-SH2), acatalytic domain (PTP), and a C-terminal tail. The two SH2 domainscontrol the subcellular localization and functional regulation of SHP2.The molecule exists in an inactive conformation, inhibiting its ownactivity via a binding network involving residues from both the N-SH2and PTP domains. In response to growth factor stimulation, SHP2 binds tospecific tyrosine-phosphorylated sites on docking proteins such as Gab1and Gab2 via its SH2 domains. This induces a conformational change thatresults in SHP2 activation.

Mutations in PTPN11 have been identified in several human diseases, suchas Noonan Syndrome, Leopard Syndrome, juvenile myelomonocytic leukemias,neuroblastoma, melanoma, acute myeloid leukemia and cancers of thebreast, lung and colon. SHP2 is an important downstream signalingmolecule for a variety of receptor tyrosine kinases, including thereceptors of platelet-derived growth factor (PDGF-R), fibroblast growthfactor (FGF-R) and epidermal growth factor (EGF-R). SHP2 is also animportant downstream signaling molecule for the activation of themitogen activated protein (MAP) kinase pathway which can lead to celltransformation, a prerequisite for the development of cancer. Knock-downof SHP2 significantly inhibited cell growth of lung cancer cell lineswith SHP2 mutation or EML4/ALK translocations as well as EGFR amplifiedbreast cancers and esophageal cancers. SHP2 is also activated downstreamof oncogenes in gastric carcinoma, anaplastic large-cell lymphoma andglioblastoma.

Noonan Syndrome (NS) and Leopard Syndrome (LS)—PTPN11 mutations cause LS(multiple lentigenes, electrocardiographic conduction abnormalities,ocular hypertelorism, pulmonic stenosis, abnormal genitalia, retardationof growth, sensorineural deafness) and NS (congenital anomaliesincluding cardiac defects, craniofacial abnormalities and shortstature). Both disorders are part of a family of autosomal dominantsyndromes caused by germline mutations in components of theRAS/RAF/MEK/ERK mitogen activating protein kinase pathway, required fornormal cell growth and differentiation. Aberrant regulation of thispathway has profound effects, particularly on cardiac development,resulting in various abnormalities, including valvuloseptal defectsand/or hypertrophic cardiomyopathy (HCM). Perturbations of the MAPKsignaling pathway have been established as central to these disordersand several candidate genes along this pathway have been identified inhumans, including mutations in KRAS, NRAS, SOS1, RAF1, BRAF, MEK1, MEK2,SHOC2, and CBL. The gene most commonly mutated in NS and LS is PTPN11.Germline mutations in PTPN11 (SHP2) are found in ˜50% of the cases withNS and nearly all patients with LS that shares certain features with NS.For NS, Y62D and Y63C substitutions in the protein are largely invariantand are among the most common mutations. Both these mutations affect thecatalytically inactive conformation of SHP2 without perturbing thebinding of the phosphatase to its phosphorylated signaling partners.

Juvenile Myelomonocytic Leukemias (JMML)—Somatic mutations in PTPN11(SHP2) occur in about 35% of the patients with JMML, a childhoodmyeloproliferative disorder (MPD). These gain-of-function mutations aretypically point mutations in the N-SH2 domain or in the phosphatasedomain, which prevent self-inhibition between the catalytic domain andthe N-SH2 domain, resulting in SHP2 activity.

Acute Myeloid Leukemia—PTPN11 mutations have been identified in: ˜10% ofpediatric acute leukemias, such as myelodysplastic syndrome (MDS); ˜7%of B cell acute lymphoblastic leukemia (B-ALL); and ˜4% of acute myeloidleukemia (AML).

NS and leukemia mutations cause changes in amino acids located at theinterface formed by the N-SH2 and PTP domains in the self-inhibited SHP2conformation, disrupting the inhibitory intramolecular interaction,leading to hyperactivity of the catalytic domain.

SHP2 acts as a positive regulator in receptor tyrosine kinase (RTK)signaling. Cancers containing RTK alterations (EGFR^(amp), Her2^(amp),FGFR^(amp), Met^(amp), translocated/activated RTK, i.e. ALK, BCR/ABL)include Esophageal, Breast, Lung, Colon, Gastric, Glioma, Head and Neckcancers.

Esophageal cancer (or oesophageal cancer) is a malignancy of theesophagus. There are various subtypes, primarily squamous cell cancer(<50%) and adenocarcinoma. There is a high rate of RTK expression inesophageal adenocarcinoma and squamous cell cancer. A SHP2 inhibitor ofthe invention can, therefore, be employed for innovative treatmentstrategies.

Breast cancer is a major type of cancer and a leading cause of death inwomen, where patients develop resistance to current drugs. There arefour major subtypes of breast cancers including luminal A, luminal B,Her2 like, and triple negative/Basal-like. Triple negative breast cancer(TNBC) is an aggressive breast cancer lacking specific targeted therapy.Epidermal growth factor receptor I (EGFR) has emerged as a promisingtarget in TNBC. Inhibition of Her2 as well as EGFR via SHP2 may be apromising therapy in breast cancer.

Lung Cancer—NSCLC is currently a major cause of cancer-relatedmortality. accounting for about 85% of lung cancers (predominantlyadenocarcinomas and squamous cell carcinomas). Although cytotoxicchemotherapy remains an important part of treatment, targeted therapiesbased on genetic alterations such as EGFR and ALK in the tumor are morelikely to benefit from a targeted therapy.

Colon Cancer—Approximately 30% to 50% of colorectal tumors are known tohave a mutated (abnormal) KRAS, and BRAF mutations occur in 10 to 15% ofcolorectal cancers. For a subset of patients whose colorectal tumorshave been demonstrated to over express EGFR, these patients exhibit afavorable clinical response to anti-EGFR therapy.

Gastic Cancer is one of the most prevalent cancer types. Aberrantexpression of tyrosine kinases, as reflected by the aberrant tyrosinephosphorylation in gastric cancer cells, is known in the art. Threereceptor-tyrosine kinases, c-met (HGF receptor), FGF receptor 2, anderbB2/neu are frequently amplified in gastric carcinomas. Thus,subversion of different signal pathways may contribute to theprogression of different types of gastric cancers.

Neuroblastoma is a pediatric tumor of the developing sympathetic nervoussystem, accounting for about 8% of childhood cancers. Genomicalterations of the anaplastic lymphoma kinase (ALK) gene have beenpostulated to contribute to neuroblastoma pathogenesis.

Squamous-cell carcinoma of the head and neck (SCCHN). High levels ofEGFR expression are correlated with poor prognosis and resistance toradiation therapy in a variety of cancers, mostly in squamous-cellcarcinoma of the head and neck (SCCHN). Blocking of the EGFR signalingresults in inhibition of the stimulation of the receptor, cellproliferation, and reduced invasiveness and metastases. The EGFR is,therefore, a prime target for new anticancer therapy in SCCHN.

The present invention relates to compounds capable of inhibiting theactivity of SHP2. The invention further provides a process for thepreparation of compounds of the invention and pharmaceuticalpreparations comprising such compounds. Another aspect of the presentinvention relates to a method of treating SHP2-mediated disorderscomprising the step of administering to a patient in need thereof atherapeutically effective amount of a compound of formula I as definedin the Summary of the Invention.

In certain embodiments, the present invention relates to theaforementioned method, wherein said SHP2-mediated disorders are cancersselected from, but not limited to: JMML; AML; MDS; B-ALL; neuroblastoma;esophageal; breast cancer; lung cancer; colon cancer; Gastric cancer,Head and Neck cancer.

The compounds of the present invention may also be useful in thetreatment of other diseases or conditions related to the aberrantactivity of SHP2. Thus, as a further aspect, the invention relates to amethod of treatment of a disorder selected from: NS; LS; JMML; AML; MDS;B-ALL; neuroblastoma; esophageal; breast cancer; lung cancer; coloncancer; gastric cancer; head and neck cancer.

A SHP2 inhibitor of the present invention may be usefully combined withanother pharmacologically active compound, or with two or more otherpharmacologically active compounds, particularly in the treatment ofcancer. For example, a compound of the formula (I), or apharmaceutically acceptable salt thereof, as defined above, may beadministered simultaneously, sequentially or separately in combinationwith one or more agents selected from chemotherapy agents, for example,mitotic inhibitors such as a taxane, a vinca alkaloid, paclitaxel,docetaxel, vincristine, vinblastine, vinorelbine or vinflunine, andother anticancer agents, e.g. cisplatin, 5-fluorouracil or5-fluoro-2-4(1H,3H)-pyrimidinedione (5FU), flutamide or gemcitabine.

Such combinations may offer significant advantages, includingsynergistic activity, in therapy.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is administeredparenterally.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is administeredintramuscularly, intravenously, subcutaneously, orally, pulmonary,intrathecally, topically or intranasally.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is administeredsystemically.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient is a mammal.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient is a primate.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient is a human.

In another aspect, the present invention relates to a method of treatingan SHP2-mediated disorder, comprising the step of: administering to apatient in need thereof a therapeutically effective amount of achemothereutic agent in combination with a therapeutically effectiveamount of a compound of formula I as defined in the Summary of theInvention.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,e.g., those targeted for buccal, sublingual, and systemic absorption,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; (8) nasally; (9) pulmonary; or (10) intrathecally.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about0.1 percent to about ninety-nine percent of active ingredient,preferably from about 5 percent to about 70 percent, most preferablyfrom about 10 percent to about 30 percent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and a compoundof the present invention. In certain embodiments, an aforementionedformulation renders orally bioavailable a compound of the presentinvention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, trouches and thelike), the active ingredient is mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner.Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99% (morepreferably, 10 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, oral, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 100 mg per kilogram of body weightper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. Preferred dosing is one administrationper day.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the subject compounds, as described above,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin, lungs, or mucous membranes; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually or buccally; (6) ocularly; (7) transdermally; or(8) nasally.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

Microemulsification technology can improve bioavailability of somelipophilic (water insoluble) pharmaceutical agents. Examples includeTrimetrine (Dordunoo, S. K., et al., Drug Development and IndustrialPharmacy, 17(12), 1685-1713, 1991 and REV 5901 (Sheen, P. C., et al., JPharm Sci 80(7), 712-714, 1991). Among other things, microemulsificationprovides enhanced bioavailability by preferentially directing absorptionto the lymphatic system instead of the circulatory system, which therebybypasses the liver, and prevents destruction of the compounds in thehepatobiliary circulation.

While all suitable amphiphilic carriers are contemplated, the presentlypreferred carriers are generally those that haveGenerally-Recognized-as-Safe (GRAS) status, and that can both solubilizethe compound of the present invention and microemulsify it at a laterstage when the solution comes into a contact with a complex water phase(such as one found in human gastro-intestinal tract). Usually,amphiphilic ingredients that satisfy these requirements have HLB(hydrophilic to lipophilic balance) values of 2-20, and their structurescontain straight chain aliphatic radicals in the range of C-6 to C-20.Examples are polyethylene-glycolized fatty glycerides and polyethyleneglycols.

Commercially available amphiphilic carriers are particularlycontemplated, including Gelucire-series, Labrafil, Labrasol, orLauroglycol (all manufactured and distributed by Gattefosse Corporation,Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurateand di-laurate, Lecithin, Polysorbate 80, etc (produced and distributedby a number of companies in USA and worldwide).

Hydrophilic polymers suitable for use in the present invention are thosewhich are readily water-soluble, can be covalently attached to avesicle-forming lipid, and which are tolerated in vivo without toxiceffects (i.e., are biocompatible). Suitable polymers includepolyethylene glycol (PEG), polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolicacid copolymer, and polyvinyl alcohol. Preferred polymers are thosehaving a molecular weight of from about 100 or 120 daltons up to about5,000 or 10,000 daltons, and more preferably from about 300 daltons toabout 5,000 daltons. In a particularly preferred embodiment, the polymeris polyethyleneglycol having a molecular weight of from about 100 toabout 5,000 daltons, and more preferably having a molecular weight offrom about 300 to about 5,000 daltons. In a particularly preferredembodiment, the polymer is polyethyleneglycol of 750 daltons (PEG(750)).Polymers may also be defined by the number of monomers therein; apreferred embodiment of the present invention utilizes polymers of atleast about three monomers, such PEG polymers consisting of threemonomers (approximately 150 daltons).

Other hydrophilic polymers which may be suitable for use in the presentinvention include polyvinylpyrrolidone, polymethoxazoline,polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose.

In certain embodiments, a formulation of the present invention comprisesa biocompatible polymer selected from the group consisting ofpolyamides, polycarbonates, polyalkylenes, polymers of acrylic andmethacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, celluloses, polypropylene,polyethylenes, polystyrene, polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronicacids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8glucose units, designated by the Greek letter alpha, beta or gamma,respectively. Cyclodextrins with fewer than six glucose units are notknown to exist. The glucose units are linked by alpha-1,4-glucosidicbonds. As a consequence of the chair conformation of the sugar units,all secondary hydroxyl groups (at C-2, C-3) are located on one side ofthe ring, while all the primary hydroxyl groups at C-6 are situated onthe other side. As a result, the external faces are hydrophilic, makingthe cyclodextrins water-soluble. In contrast, the cavities of thecyclodextrins are hydrophobic, since they are lined by the hydrogen ofatoms C-3 and C-5, and by ether-like oxygens. These matrices allowcomplexation with a variety of relatively hydrophobic compounds,including, for instance, steroid compounds such as 17.beta.-estradiol(see, e.g., van Uden et al. Plant Cell Tiss. Org. Cult. 38:1-3-113(1994)). The complexation takes place by Van der Waals interactions andby hydrogen bond formation. For a general review of the chemistry ofcyclodextrins, see, Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822(1994).

The physico-chemical properties of the cyclodextrin derivatives dependstrongly on the kind and the degree of substitution. For example, theirsolubility in water ranges from insoluble (e.g.,triacetyl-beta-cyclodextrin) to 147% soluble (w/v)(G-2-beta-cyclodextrin). In addition, they are soluble in many organicsolvents. The properties of the cyclodextrins enable the control oversolubility of various formulation components by increasing or decreasingtheir solubility.

Numerous cyclodextrins and methods for their preparation have beendescribed. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259)and Gramera, et al. (U.S. Pat. No. 3,459,731) described electroneutralcyclodextrins. Other derivatives include cyclodextrins with cationicproperties [Parmeter (II), U.S. Pat. No. 3,453,257], insolublecrosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), andcyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No.3,426,011]. Among the cyclodextrin derivatives with anionic properties,carboxylic acids, phosphorous acids, phosphinous acids, phosphonicacids, phosphoric acids, thiophosphonic acids, thiosulphinic acids, andsulfonic acids have been appended to the parent cyclodextrin [see,Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrinderivatives have been described by Stella, et al. (U.S. Pat. No.5,134,127).

Liposomes consist of at least one lipid bilayer membrane enclosing anaqueous internal compartment. Liposomes may be characterized by membranetype and by size. Small unilamellar vesicles (SUVs) have a singlemembrane and typically range between 0.02 and 0.05 μm in diameter; largeunilamellar vesicles (LUVS) are typically larger than 0.05 μmOligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers and are typically larger than 0.1μm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

One aspect of the present invention relates to formulations comprisingliposomes containing a compound of the present invention, where theliposome membrane is formulated to provide a liposome with increasedcarrying capacity. Alternatively or in addition, the compound of thepresent invention may be contained within, or adsorbed onto, theliposome bilayer of the liposome. The compound of the present inventionmay be aggregated with a lipid surfactant and carried within theliposome's internal space; in these cases, the liposome membrane isformulated to resist the disruptive effects of the activeagent-surfactant aggregate.

According to one embodiment of the present invention, the lipid bilayerof a liposome contains lipids derivatized with polyethylene glycol(PEG), such that the PEG chains extend from the inner surface of thelipid bilayer into the interior space encapsulated by the liposome, andextend from the exterior of the lipid bilayer into the surroundingenvironment.

Active agents contained within liposomes of the present invention are insolubilized form. Aggregates of surfactant and active agent (such asemulsions or micelles containing the active agent of interest) may beentrapped within the interior space of liposomes according to thepresent invention. A surfactant acts to disperse and solubilize theactive agent, and may be selected from any suitable aliphatic,cycloaliphatic or aromatic surfactant, including but not limited tobiocompatible lysophosphatidylcholines (LPCs) of varying chain lengths(for example, from about C.sub.14 to about C.sub.20).Polymer-derivatized lipids such as PEG-lipids may also be utilized formicelle formation as they will act to inhibit micelle/membrane fusion,and as the addition of a polymer to surfactant molecules decreases theCMC of the surfactant and aids in micelle formation. Preferred aresurfactants with CMCs in the micromolar range; higher CMC surfactantsmay be utilized to prepare micelles entrapped within liposomes of thepresent invention, however, micelle surfactant monomers could affectliposome bilayer stability and would be a factor in designing a liposomeof a desired stability.

Liposomes according to the present invention may be prepared by any of avariety of techniques that are known in the art. See, e.g., U.S. Pat.No. 4,235,871; Published PCT applications WO 96/14057; New RRC,Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104;Lasic D D, Liposomes from physics to applications, Elsevier SciencePublishers BV, Amsterdam, 1993.

For example, liposomes of the present invention may be prepared bydiffusing a lipid derivatized with a hydrophilic polymer into preformedliposomes, such as by exposing preformed liposomes to micelles composedof lipid-grafted polymers, at lipid concentrations corresponding to thefinal mole percent of derivatized lipid which is desired in theliposome. Liposomes containing a hydrophilic polymer can also be formedby homogenization, lipid-field hydration, or extrusion techniques, asare known in the art.

In one aspect of the present invention, the liposomes are prepared tohave substantially homogeneous sizes in a selected size range. Oneeffective sizing method involves extruding an aqueous suspension of theliposomes through a series of polycarbonate membranes having a selecteduniform pore size; the pore size of the membrane will correspond roughlywith the largest sizes of liposomes produced by extrusion through thatmembrane. See e.g., U.S. Pat. No. 4,737,323 (Apr. 12, 1988).

The release characteristics of a formulation of the present inventiondepend on the encapsulating material, the concentration of encapsulateddrug, and the presence of release modifiers. For example, release can bemanipulated to be pH dependent, for example, using a pH sensitivecoating that releases only at a low pH, as in the stomach, or a higherpH, as in the intestine. An enteric coating can be used to preventrelease from occurring until after passage through the stomach. Multiplecoatings or mixtures of cyanamide encapsulated in different materialscan be used to obtain an initial release in the stomach, followed bylater release in the intestine. Release can also be manipulated byinclusion of salts or pore forming agents, which can increase wateruptake or release of drug by diffusion from the capsule. Excipientswhich modify the solubility of the drug can also be used to control therelease rate. Agents which enhance degradation of the matrix or releasefrom the matrix can also be incorporated. They can be added to the drug,added as a separate phase (i.e., as particulates), or can beco-dissolved in the polymer phase depending on the compound. In allcases the amount should be between 0.1 and thirty percent (w/w polymer).Types of degradation enhancers include inorganic salts such as ammoniumsulfate and ammonium chloride, organic acids such as citric acid,benzoic acid, and ascorbic acid, inorganic bases such as sodiumcarbonate, potassium carbonate, calcium carbonate, zinc carbonate, andzinc hydroxide, and organic bases such as protamine sulfate, spermine,choline, ethanolamine, diethanolamine, and triethanolamine andsurfactants such as Tween® and Pluronic®. Pore forming agents which addmicrostructure to the matrices (i.e., water soluble compounds such asinorganic salts and sugars) are added as particulates. The range shouldbe between one and thirty percent (w/w polymer).

Uptake can also be manipulated by altering residence time of theparticles in the gut. This can be achieved, for example, by coating theparticle with, or selecting as the encapsulating material, a mucosaladhesive polymer. Examples include most polymers with free carboxylgroups, such as chitosan, celluloses, and especially polyacrylates (asused herein, polyacrylates refers to polymers including acrylate groupsand modified acrylate groups such as cyanoacrylates and methacrylates).

Pharmaceutical Combinations

The invention especially relates to the use of a compound of the formulaI (or a pharmaceutical composition comprising a compound of the formulaI) in the treatment of one or more of the diseases mentioned herein;wherein the response to treatment is beneficial as demonstrated, forexample, by the partial or complete removal of one or more of thesymptoms of the disease up to complete cure or remission.

A compound of formula (I) can also be used in combination with thefollowing compounds:

BCR-ABL inhibitors: Imatinib (Gleevec®); Inilotinib hydrochloride;Nilotinib (Tasigna®); Dasatinib (BMS-345825); Bosutinib (SKI-606);Ponatinib (AP24534); Bafetinib (INNO406); Danusertib (PHA-739358),AT9283 (CAS 1133385-83-7); Saracatinib (AZD0530); andN-[2-[(1S,4R)-6-[[4-(Cyclobutylamino)-5-(trifluoromethyl)-2-pyrimidinyl]amino]-1,2,3,4-tetrahydronaphthalen-1,4-imin-9-yl]-2-oxoethyl]-acetamide(PF-03814735, CAS 942487-16-3); and LGX818.

ALK inhibitors: PF-2341066 (XALKORI®; crizotinib);5-chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)pyrimidine-2,4-diamine;GSK1838705A; and CH5424802.

BRAF inhibitors: Vemurafanib (PLX4032); and Dabrafenib.

FLT3 inhibitors—sunitinib malate (sold under the tradename Sutent® byPfizer); and PKC412 (midostaurin).

MEK Inhibitors—trametinib.

Vascular Endothelial Growth Factor (VEGF) receptor inhibitors:Bevacizumab (sold under the trademark Avastin® by Genentech/Roche),axitinib,(N-methyl-2-[[3-[(E)-2-pyridin-2-ylethenyl]-1H-indazol-6-yl]sulfanyl]benzamide,also known as AG013736, and described in PCT Publication No. WO01/002369), Brivanib Alaninate((S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate,also known as BMS-582664), motesanib(N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide,and described in PCT Publication No. WO 02/066470), pasireotide (alsoknown as SOM230, and described in PCT Publication No. WO 02/010192),sorafenib (sold under the tradename Nexavar®);

HER2 receptor inhibitors: Trastuzumab (sold under the trademarkHerceptin® by Genentech/Roche), neratinib (also known as HKI-272,(2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide,and described PCT Publication No. WO 05/028443), lapatinib or lapatinibditosylate (sold under the trademark Tykerb® by GlaxoSmithKline);

CD20 antibodies: Rituximab (sold under the trademarks Riuxan® andMabThera® by Genentech/Roche), tositumomab (sold under the trademarksBexxar® by GlaxoSmithKline), ofatumumab (sold under the trademarkArzerra® by GlaxoSmithKline);

Tyrosine kinase inhibitors: Erlotinib hydrochloride (sold under thetrademark Tarceva® by Genentech/Roche), Linifanib(N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N′-(2-fluoro-5-methylphenyl)urea,also known as ABT 869, available from Genentech), sunitinib malate (soldunder the tradename Sutent® by Pfizer), bosutinib(4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy]quinoline-3-carbonitrile,also known as SKI-606, and described in U.S. Pat. No. 6,780,996),dasatinib (sold under the tradename Sprycel® by Bristol-Myers Squibb),armala (also known as pazopanib, sold under the tradename Votrient® byGlaxoSmithKline), imatinib and imatinib mesylate (sold under thetradenames Gilvec® and Gleevec® by Novartis);

DNA Synthesis inhibitors: Capecitabine (sold under the trademark Xeloda®by Roche), gemcitabine hydrochloride (sold under the trademark Gemzar®by Eli Lilly and Company), nelarabine((2R,3S,4R,5R)-2-(2-amino-6-methoxy-purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol,sold under the tradenames Arranon® and Atriance® by GlaxoSmithKline);

Antineoplastic agents: oxaliplatin (sold under the tradename Eloxatin®ay Sanofi-Aventis and described in U.S. Pat. No. 4,169,846);

Epidermal growth factor receptor (EGFR) inhibitors: Gefitnib (sold underthe tradename Iressa®),N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3″S″)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide,sold under the tradename Tovok® by Boehringer Ingelheim), cetuximab(sold under the tradename Erbitux® by Bristol-Myers Squibb), panitumumab(sold under the tradename Vectibix® by Amgen);

HER dimerization inhibitors: Pertuzumab (sold under the trademarkOmnitarg®, by Genentech);

Human Granulocyte colony-stimulating factor (G-CSF) modulators:Filgrastim (sold under the tradename Neupogen® by Amgen);

Immunomodulators: Afutuzumab (available from Roche®), pegfilgrastim(sold under the tradename Neulasta® by Amgen), lenalidomide (also knownas CC-5013, sold under the tradename Revlimid®), thalidomide (sold underthe tradename Thalomid®);

CD40 inhibitors: Dacetuzumab (also known as SGN-40 or huS2C6, availablefrom Seattle Genetics, Inc);

Pro-apoptotic receptor agonists (PARAs): Dulanermin (also known asAMG-951, available from Amgen/Genentech);

Hedgehog antagonists:2-chloro-N-[4-chloro-3-(2-pyridinyl)phenyl]-4-(methylsulfonyl)-benzamide(also known as GDC-0449, and described in PCT Publication No. WO06/028958);

PI3K inhibitors:4-[2-(1H-Indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine(also known as GDC 0941 and described in PCT Publication Nos. WO09/036082 and WO 09/055730),2-Methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile(also known as BEZ 235 or NVP-BEZ 235, and described in PCT PublicationNo. WO 06/122806);

Phospholipase A2 inhibitors: Anagrelide (sold under the tradenameAgrylin®);

BCL-2 inhibitors:4-[4-[[2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(4-morpholinyl)-1-[(phenylthio)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl]benzamide(also known as ABT-263 and described in PCT Publication No. WO09/155386);

Mitogen-activated protein kinase kinase (MEK) inhibitors: XL-518 (CasNo. 1029872-29-4, available from ACC Corp.);

Aromatase inhibitors: Exemestane (sold under the trademark Aromasin® byPfizer), letrozole (sold under the tradename Femara® by Novartis),anastrozole (sold under the tradename Arimidex®);

Topoisomerase I inhibitors: Irinotecan (sold under the trademarkCamptosar® by Pfizer), topotecan hydrochloride (sold under the tradenameHycamtin® by GlaxoSmithKline);

Topoisomerase II inhibitors: etoposide (also known as VP-16 andEtoposide phosphate, sold under the tradenames Toposar®, VePesid® andEtopophos®), teniposide (also known as VM-26, sold under the tradenameVumon®);

mTOR inhibitors: Temsirolimus (sold under the tradename Torisel® byPfizer), ridaforolimus (formally known as deferolimus,(1R,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383), everolimus (sold under the tradenameAfinitor® by Novartis);

Osteoclastic bone resorption inhibitors:1-Hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acid monohydrate(sold under the tradename Zometa® by Novartis);

CD33 Antibody Drug Conjugates: Gemtuzumab ozogamicin (sold under thetradename Mylotarg® by Pfizer/Wyeth);

CD22 Antibody Drug Conjugates: Inotuzumab ozogamicin (also referred toas CMC-544 and WAY-207294, available from Hangzhou Sage Chemical Co.,Ltd.);

CD20 Antibody Drug Conjugates: Ibritumomab tiuxetan (sold under thetradename Zevalin®);

Somatostain analogs: octreotide (also known as octreotide acetate, soldunder the tradenames Sandostatin® and Sandostatin LAR®);

Synthetic Interleukin-11 (IL-11): oprelvekin (sold under the tradenameNeumega® by Pfizer/Wyeth);

Synthetic erythropoietin: Darbepoetin alfa (sold under the tradenameAranesp® by Amgen);

Receptor Activator for Nuclear Factor κB (RANK) inhibitors: Denosumab(sold under the tradename Prolia® by Amgen);

Thrombopoietin mimetic peptibodies: Romiplostim (sold under thetradename Nplate® by Amgen;

Cell growth stimulators: Palifermin (sold under the tradename Kepivance®by Amgen);

Anti-Insulin-like Growth Factor-1 receptor (IGF-1R) antibodies:Figitumumab (also known as CP-751,871, available from ACC Corp),robatumumab (CAS No. 934235-44-6);

Anti-CS1 antibodies: Elotuzumab (HuLuc63, CAS No. 915296-00-3);

CD52 antibodies: Alemtuzumab (sold under the tradename Campath®);

CTLA-4 inhibitors: Tremelimumab (IgG2 monoclonal antibody available fromPfizer, formerly known as ticilimumab, CP-675,206), ipilimumab (CTLA-4antibody, also known as MDX-010, CAS No. 477202-00-9);

Histone deacetylase inhibitors (HDI): Voninostat (sold under thetradename Zolinza® by Merck);

Alkylating agents: Temozolomide (sold under the tradenames Temodar® andTemodal® by Schering-Plough/Merck), dactinomycin (also known asactinomycin-D and sold under the tradename Cosmegen®), melphalan (alsoknown as L-PAM, L-sarcolysin, and phenylalanine mustard, sold under thetradename Alkeran®), altretamine (also known as hexamethylmelamine(HMM), sold under the tradename Hexalen®), carmustine (sold under thetradename BiCNU®), bendamustine (sold under the tradename Treanda®),busulfan (sold under the tradenames Busulfex® and Myleran®), carboplatin(sold under the tradename Paraplatin®), lomustine (also known as CCNU,sold under the tradename CeeNU®), cisplatin (also known as CDDP, soldunder the tradenames Platinol® and Platinol®-AQ), chlorambucil (soldunder the tradename Leukeran®), cyclophosphamide (sold under thetradenames Cytoxan® and Neosar®), dacarbazine (also known as DTIC, DICand imidazole carboxamide, sold under the tradename DTIC-Dome®),altretamine (also known as hexamethylmelamine (HMM) sold under thetradename Hexalen®), ifosfamide (sold under the tradename Ifex®),procarbazine (sold under the tradename Matulane®), mechlorethamine (alsoknown as nitrogen mustard, mustine and mechloroethamine hydrochloride,sold under the tradename Mustargen®), streptozocin (sold under thetradename Zanosar®), thiotepa (also known as thiophosphoamide, TESPA andTSPA, sold under the tradename Thioplex®;

Biologic response modifiers: bacillus calmette-guerin (sold under thetradenames theraCys® and TICE® BCG), denileukin diftitox (sold under thetradename Ontak®);

Anti-tumor antibiotics: doxorubicin (sold under the tradenamesAdriamycin® and Rubex®), bleomycin (sold under the tradename lenoxane®),daunorubicin (also known as dauorubicin hydrochloride, daunomycin, andrubidomycin hydrochloride, sold under the tradename Cerubidine®),daunorubicin liposomal (daunorubicin citrate liposome, sold under thetradename DaunoXome®), mitoxantrone (also known as DHAD, sold under thetradename Novantrone®), epirubicin (sold under the tradename Ellence™),idarubicin (sold under the tradenames Idamycin®, Idamycin PFS®),mitomycin C (sold under the tradename Mutamycin®);

Anti-microtubule agents: Estramustine (sold under the tradename Emcyl®);

Cathepsin K inhibitors: Odanacatib (also know as MK-0822,N-(1-cyanocyclopropyl)-4-fluoro-N²-{(1S)-2,2,2-trifluoro-1-[4′-(methylsulfonyl)biphenyl-4-yl]ethyl}-L-leucinamide,available from Lanzhou Chon Chemicals, ACC Corp., and ChemieTek, anddescribed in PCT Publication no. WO 03/075836);

Epothilone B analogs: Ixabepilone (sold under the tradename Lxempra® byBristol-Myers Squibb);

Heat Shock Protein (HSP) inhibitors: Tanespimycin(17-allylamino-17-demethoxygeldanamycin, also known as KOS-953 and17-AAG, available from SIGMA, and described in U.S. Pat. No. 4,261,989);

TpoR agonists: Eltrombopag (sold under the tradenames Promacta® andRevolade® by GlaxoSmithKline);

Anti-mitotic agents: Docetaxel (sold under the tradename Taxotere® bySanofi-Aventis);

Adrenal steroid inhibitors: aminoglutethimide (sold under the tradenameCytadren®);

Anti-androgens: Nilutamide (sold under the tradenames Nilandron® andAnandron®), bicalutamide (sold under tradename Casodex®), flutamide(sold under the tradename Fulexin™);

Androgens: Fluoxymesterone (sold under the tradename Halotestin®);

Proteasome inhibitors: Bortezomib (sold under the tradename Velcade®);

CDK1 inhibitors: Alvocidib (also known as flovopirdol or HMR-1275,2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone,and described in U.S. Pat. No. 5,621,002);

Gonadotropin-releasing hormone (GnRH) receptor agonists: Leuprolide orleuprolide acetate (sold under the tradenames Viadure® by Bayer AG,Eligard® by Sanofi-Aventis and Lupron® by Abbott Lab);

Taxane anti-neoplastic agents: Cabazitaxel(1-hydroxy-7β,10β-dimethoxy-9-oxo-5β,20-epoxytax-11-ene-2α,4,13α-triyl-4-acetate-2-benzoate-13-[(2R,3S)-3-{[(tert-butoxy)carbonyl]amino}-2-hydroxy-3-phenylpropanoate),larotaxel((2α,3ξ,4α,5β,7α,10β,13α)-4,10-bis(acetyloxy)-13-({(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropanoyl}oxy)-1-hydroxy-9-oxo-5,20-epoxy-7,19-cyclotax-11-en-2-ylbenzoate);

5HT1a receptor agonists: Xaliproden (also known as SR57746,1-[2-(2-naphthyl)ethyl]-4-[3-(trifluoromethyl)phenyl]-1,2,3,6-tetrahydropyridine,and described in U.S. Pat. No. 5,266,573);

HPC vaccines: Cervarix® sold by GlaxoSmithKline, Gardasil® sold byMerck;

Iron Chelating agents: Deferasinox (sold under the tradename Exjade® byNovartis);

Anti-metabolites: Claribine (2-chlorodeoxyadenosine, sold under thetradename leustatin®), 5-fluorouracil (sold under the tradenameAdrucil®), 6-thioguanine (sold under the tradename Purinethol®),pemetrexed (sold under the tradename Alimta®), cytarabine (also known asarabinosylcytosine (Ara-C), sold under the tradename Cytosar-U®),cytarabine liposomal (also known as Liposomal Ara-C, sold under thetradename DepoCyt™), decitabine (sold under the tradename Dacogen®),hydroxyurea (sold under the tradenames Hydrea®, Droxia™ and Mylocel™),fludarabine (sold under the tradename Fludara®), floxuridine (sold underthe tradename FUDR®), cladribine (also known as 2-chlorodeoxyadenosine(2-CdA) sold under the tradename Leustatin™), methotrexate (also knownas amethopterin, methotrexate sodim (MTX), sold under the tradenamesRheumatrex® and Trexall™), pentostatin (sold under the tradenameNipent®);

Bisphosphonates: Pamidronate (sold under the tradename Aredia®),zoledronic acid (sold under the tradename Zometa®);

Demethylating agents: 5-azacitidine (sold under the tradename Vidaza®),decitabine (sold under the tradename Dacogen®);

Plant Alkaloids: Paclitaxel protein-bound (sold under the tradenameAbraxane®), vinblastine (also known as vinblastine sulfate,vincaleukoblastine and VLB, sold under the tradenames Alkaban-AQ® andVelban®), vincristine (also known as vincristine sulfate, LCR, and VCR,sold under the tradenames Oncovin® and Vincasar Pfs®), vinorelbine (soldunder the tradename Navelbine®), paclitaxel (sold under the tradenamesTaxol and Onxal™);

Retinoids: Alitretinoin (sold under the tradename Panretin®), tretinoin(all-trans retinoic acid, also known as ATRA, sold under the tradenameVesanoid®), Isotretinoin (13-cis-retinoic acid, sold under thetradenames Accutane®, Amnesteem®, Claravis®, Clarus®, Decutan®,Isotane®, Izotech®, Oratane®, Isotret®, and Sotret®), bexarotene (soldunder the tradename Targretin®);

Glucocorticosteroids: Hydrocortisone (also known as cortisone,hydrocortisone sodium succinate, hydrocortisone sodium phosphate, andsold under the tradenames Ala-Cort®, Hydrocortisone Phosphate,Solu-Cortef®, Hydrocort Acetate® and Lanacort®), dexamethazone((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one),prednisolone (sold under the tradenames Delta-Cortel®, Orapred®,Pediapred® and Prelone®), prednisone (sold under the tradenamesDeltasone®, Liquid Red®, Meticorten® and Orasone®), methylprednisolone(also known as 6-Methylprednisolone, Methylprednisolone Acetate,Methylprednisolone Sodium Succinate, sold under the tradenamesDuralone®, Medralone®, Medrol®, M-Prednisol® and Solu-Medrol®);

Cytokines: interleukin-2 (also known as aldesleukin and IL-2, sold underthe tradename Proleukin®), interleukin-11 (also known as oprevelkin,sold under the tradename Neumega®), alpha interferon alfa (also known asIFN-alpha, sold under the tradenames Intron® A, and Roferon-A®);

Estrogen receptor downregulators: Fulvestrant (sold under the tradenameFaslodex®);

Anti-estrogens: tamoxifen (sold under the tradename Novaldex®);

Toremifene (sold under the tradename Fareston®);

Selective estrogen receptor modulators (SERMs): Raloxifene (sold underthe tradename Evista®);

Leutinizing hormone releasing hormone (LHRH) agonists: Goserelin (soldunder the tradename Zoladex®);

Progesterones: megestrol (also known as megestrol acetate, sold underthe tradename Megace®);

Miscellaneous cytotoxic agents: Arsenic trioxide (sold under thetradename Trisenox®), asparaginase (also known as L-asparaginase,Erwinia L-asparaginase, sold under the tradenames Elspar® andKidrolase®);

A compound of formula (I) can also be used in combination with thefollowing adjunct therapies:

Anti-nausea drugs: NK-1 receptor antagonists: Casopitant (sold under thetradenames Rezonic® and Zunrisa® by GlaxoSmithKline); and

Cytoprotective agents: Amifostine (sold under the tradename Ethyol®),leucovorin (also known as calcium leucovorin, citrovorum factor andfolinic acid).

None of the quotations of references made within the present disclosureis to be understood as an admission that the references cited are priorart that would negatively affect the patentability of the presentinvention.

Processes for Making Compounds of the Invention

The present invention also includes processes for the preparation ofcompounds of the invention. In the reactions described, it can benecessary to protect reactive functional groups, for example hydroxy,amino, imino, thio or carboxy groups, where these are desired in thefinal product, to avoid their unwanted participation in the reactions.Conventional protecting groups can be used in accordance with standardpractice, for example, see T. W. Greene and P. G. M. Wuts in “ProtectiveGroups in Organic Chemistry”, John Wiley and Sons, 1991.

Compounds of Formula I, can be prepared by proceeding as in thefollowing Reaction Scheme I:

m, p, Y₁, Y₂, Y₃, R₁, R_(2a), R_(2b), R_(3a), R_(3b), R_(4a), R_(4b),R_(5a) and R_(5b) are as defined for Formula I in the Summary of theInvention and Q is a halogen (like bromine) or a thiol, boronate orstannate which reacts with a halogen on compound 5 and X is a reactivegroup which reacts with Q (such as a boronate, stannane, alcohol, thiol,halogen, and the like). Compound 4 may be prepared by reacting compound2 with compound 3 through a reaction under suitable acid or baseconditions in the presence or absence of a transition metal underambient temperature, or under thermal or microwave conditions.Alternatively, the halogen of compound 2 may be replaced by otherhalogens or suitable activating groups such as triflates, mesylates,tosylates, nonaflates, boronates, organostannanes, organosilyls,organozincs, lithium, magnesium, and the like.

A compound of formula I can be prepared by reacting compound 4 with asuitable coupling partner (e.g. compound 5) depending on X. For example,compound 5 is shown in reaction scheme I as a substituted phenyl grouplinked via X. Alternatively, compound 5 could be aryl-alcohol,aryl-thio, aryl-boronate, aryl-stannate, heteroaryl-alcohol,heteroaryl-thiol, aryl-boronate, aryl-stannane, olefin, or otheraryl-metals or heteroaryl-metals, and the like. The coupling partnersmay also be substituted. This reaction may be conducted under suitableacid or base conditions, in the presence or absence of a transitionmetal such as palladium, under ambient temperature, or under thermal ormicrowave conditions. Other halogens or suitable activating groups(e.g., triflates, mesylates, tosylates, and nonaflates) may be used inplace of Br for these transformations.

Alternatively, the coupling partners could be reversed and compound 2may be derivatized to a stannane, boronate, organo-zinc, organo-lithium,organo-magnesium, organo-silicon, organo-cuprate and coupled with asuitable aryl-halide, heteroaryl-halide, olefin or suitable reactivefunctional group (e.g., triflates, mesylates, tosylates and nonaflates),and the like.

These reactions may be conducted in the order described or in reverseorder, under a variety of solvents, temperatures, pressures, and undersuitable atmospheres. The reactions may be conducted under acid, base,and or transition metal conditions.

Detailed examples of the synthesis of compounds of Formula I can befound in the Examples, infra.

Additional Processes for Making Compounds of the Invention

A compound of the invention can be prepared as a pharmaceuticallyacceptable acid addition salt by reacting the free base form of thecompound with a pharmaceutically acceptable inorganic or organic acid.Alternatively, a pharmaceutically acceptable base addition salt of acompound of the invention can be prepared by reacting the free acid formof the compound with a pharmaceutically acceptable inorganic or organicbase.

Compounds of the formula I can also be modified by appending appropriatefunctionalities to enhance selective biological properties.Modifications of this kind are known in the art and include those thatincrease penetration into a given biological system (e.g. blood,lymphatic system, central nervous system, testis), increasebioavailability, increase solubility to allow parenteral administration(e.g. injection, infusion), alter metabolism and/or alter the rate ofsecretion. Examples of this type of modifications include but are notlimited to esterification, e.g. with polyethylene glycols,derivatisation with pivaloyloxy or fatty acid substituents, conversionto carbamates, hydroxylation of aromatic rings and heteroatomsubstitution in aromatic rings. Whereever compounds of the formula I,and/or N-oxides, tautomers and/or (preferably pharmaceuticallyacceptable) salts thereof are mentioned, this comprises such modifiedformulae, while preferably the molecules of the formula I, theirN-oxides, their tautomers and/or their salts are meant.

Alternatively, the salt forms of the compounds of the invention can beprepared using salts of the starting materials or intermediates. In viewof the close relationship between the novel compounds of the formula Iin free form and those in the form of their salts, including those saltsthat can be used as intermediates, for example in the purification oridentification of the novel compounds, any reference to the compounds ora compound of the formula I hereinbefore and hereinafter is to beunderstood as referring to the compound in free form and/or also to oneor more salts thereof, as appropriate and expedient, as well as to oneor more solvates, e.g. hydrates.

Salts are formed, for example, as acid addition salts, preferably withorganic or inorganic acids, from compounds of formula I with a basicnitrogen atom, especially the pharmaceutically acceptable salts.Suitable inorganic acids are, for example, halogen acids, such ashydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organicacids are, for example, carboxylic, phosphonic, sulfonic or sulfamicacids, for example acetic acid, propionic acid, octanoic acid, decanoicacid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid,succinic acid, malonic acid, adipic acid, pimelic acid, suberic acid,azelaic acid, malic acid, tartaric acid, citric acid, amino acids, suchas glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylicacid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalicacid, phenylacetic acid, mandelic acid, cinnamic acid, methane- orethane-sulfonic acid, 2-hydroxyethanesulfonic acid,ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-toluenesulfonicacid, 2-naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, 2- or3-methylbenzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid,dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- orN-propyl-sulfamic acid, or other organic protonic acids, such asascorbic acid.

For isolation or purification purposes it is also possible to usepharmaceutically unacceptable salts, for example picrates orperchlorates. For therapeutic use, only pharmaceutically acceptablesalts or free compounds are employed (where applicable in the form ofpharmaceutical preparations), and these are therefore preferred.

The free acid or free base forms of the compounds of the invention canbe prepared from the corresponding base addition salt or acid additionsalt from, respectively. For example a compound of the invention in anacid addition salt form can be converted to the corresponding free baseby treating with a suitable base (e.g., ammonium hydroxide solution,sodium hydroxide, and the like). A compound of the invention in a baseaddition salt form can be converted to the corresponding free acid bytreating with a suitable acid (e.g., hydrochloric acid, etc.).

Compounds of the invention in unoxidized form can be prepared fromN-oxides of compounds of the invention by treating with a reducing agent(e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride,sodium borohydride, phosphorus trichloride, tribromide, or the like) ina suitable inert organic solvent (e.g. acetonitrile, ethanol, aqueousdioxane, or the like) at 0 to 80° C.

Prodrug derivatives of the compounds of the invention can be prepared bymethods known to those of ordinary skill in the art (e.g., for furtherdetails see Saulnier et al., (1994), Bioorganic and Medicinal ChemistryLetters, Vol. 4, p. 1985). For example, appropriate prodrugs can beprepared by reacting a non-derivatized compound of the invention with asuitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate,para-nitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the invention can be made bymeans known to those of ordinary skill in the art. A detaileddescription of techniques applicable to the creation of protectinggroups and their removal can be found in T. W. Greene, “ProtectingGroups in Organic Chemistry”, 3^(rd) edition, John Wiley and Sons, Inc.,1999.

Compounds of the present invention can be conveniently prepared, orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxin, tetrahydrofuranor methanol.

Compounds of the invention can be prepared as their individualstereoisomers by reacting a racemic mixture of the compound with anoptically active resolving agent to form a pair of diastereoisomericcompounds, separating the diastereomers and recovering the opticallypure enantiomers. While resolution of enantiomers can be carried outusing covalent diastereomeric derivatives of the compounds of theinvention, dissociable complexes are preferred (e.g., crystallinediastereomeric salts). Diastereomers have distinct physical properties(e.g., melting points, boiling points, solubilities, reactivity, etc.)and can be readily separated by taking advantage of thesedissimilarities. The diastereomers can be separated by chromatography,or preferably, by separation/resolution techniques based upondifferences in solubility. The optically pure enantiomer is thenrecovered, along with the resolving agent, by any practical means thatwould not result in racemization. A more detailed description of thetechniques applicable to the resolution of stereoisomers of compoundsfrom their racemic mixture can be found in Jean Jacques, Andre Collet,Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John WileyAnd Sons, Inc., 1981.

In summary, the compounds of Formula I can be made by a process, whichinvolves:

(a) that of reaction scheme I; and

(b) optionally converting a compound of the invention into apharmaceutically acceptable salt;

(c) optionally converting a salt form of a compound of the invention toa non-salt form;

(d) optionally converting an unoxidized form of a compound of theinvention into a pharmaceutically acceptable N-oxide;

(e) optionally converting an N-oxide form of a compound of the inventionto its unoxidized form;

(f) optionally resolving an individual isomer of a compound of theinvention from a mixture of isomers;

(g) optionally converting a non-derivatized compound of the inventioninto a pharmaceutically acceptable prodrug derivative; and

(h) optionally converting a prodrug derivative of a compound of theinvention to its non-derivatized form.

Insofar as the production of the starting materials is not particularlydescribed, the compounds are known or can be prepared analogously tomethods known in the art or as disclosed in the Examples hereinafter.

One of skill in the art will appreciate that the above transformationsare only representative of methods for preparation of the compounds ofthe present invention, and that other well known methods can similarlybe used.

EXAMPLES

The following examples and intermediates serve to illustrate theinvention without limiting the scope thereof. Some abbreviations used inthe examples are as follows: acetic acid (AcOH); triethylamine (TEA);tetrahydrofuran (THF); aqueous (aq.); atmosphere (atm.);2,2′-bis-diphenylphosphanyl-[1,1′]binaphthalenyl (BINAP);4-dimethylaminopyridine (DMAP); tert-butoxycarbonyl (Boc);1,1-carbonyldiimidazole (CDI); di-tert-butyl dicarbonate (BOC₂O);benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP); dichloromethane (DCM); diethyl ether (Et₂O);p-toluene sulfonic acid (PTSA); ethyl acetate (EtOAc); ethanol (EtOH);lithium bis(trimethylsilyl)amide (LHMDS); diisopropyl azodicarboxylate(DIAD); N,N-diisopropyl-ethylamine (DIEA or DIPEA);N,N-dimethylformamide (DMF); dimethyl sulfoxide (DMSO);diphenylphosphoryl azide (DPPA); hour(s) (h);2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU); High Performance Liquid Chromatography(HPLC); lithium aluminium hydride (LAH); liquid chromatography coupledwith mass spectrometry (LCMS); lithium diisopropylamide (LDA); methanol(MeOH); milliliter(s) (mL); minute(s) (min); microwave (MW);n-butyllithium (n-BuLi);1,1-bis(diphenylphosphino)-ferrocenedichloropalladium (II)(PdCl₂(dppf)); tris(dibenzylideneacetone)dipalladium (0) (Pd₂(dba)₃);dichlorobis(triphenylphosphine)palladium (II) (PdCl₂(PPh₃)₂); roomtemperature (RT); trifluoroacetic acid (TFA); tetrahydrofuran (THF);thin layer chromatography (TLC); retention time (t_(R)); &4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantophos).

Intermediate 12-(2,3-dichloro-5-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

Step a: A solution of 1-bromo-3-chloro-5-methoxybenzene (835 mg, 3.77mmol), and 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione (299 mg, 1.29mmol) in DMF (18 mL) was stirred 16 h under N₂ and 50° C. After coolingto RT, the reaction mixture was diluted with aqueous NH₄Cl and extractedwith Et₂O. The combined organic phases were dried over MgSO₄, filtered,concentrated, and the resulting residue was purified by silicachromatography (0 to 25% gradient of EtOAc/heptane) to1-bromo-2,3-dichloro-5-methoxybenzene (720 mg, 2.81 mmol). ¹H NMR (400MHz, CHLOROFORM-d) δ ppm 7.14 (d, J=2.76 Hz, 1 H), 7.01 (d, J=3.01 Hz, 1H), 3.81 (s, 3 H).

Step b: A suspension of 1-bromo-2,3-dichloro-5-methoxybenzene (710 mg,2.77 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane)(775 mg, 3.05 mmol), potassium acetate (817 mg, 8.32 mmol), andPdCl₂(dppf) (101 mg, 0.139 mmol) in dioxane (5.5 mL) was stirred in amicrowave reactor for 1 h at 100° C. to give the title compound assolution in dioxane that was used directly in Suzuki-Miyaura couplings.

Intermediate 2 tert-butyl(1-(4-amino-5-bromopyrimidin-2-yl)-4-methylpiperidin-4-yl)carbamate

A solution of 5-bromo-2-chloropyrimidin-4-amine (650 mg, 3.12 mmol),tert-butyl (4-methylpiperidin-4-yl)carbamate (835 mg, 3.90 mmol), and4-methylmorpholine (411 μL, 3.74 mmol) in NMP (6.25 mL) was stirred in amicrowave reactor for 90 min at 130° C. The resulting residue was pouredinto water (100 mL) and was stirred at RT for 5 min. The solid formedwas filtered off and dried under high vacuum for 16 h to give tert-butyl(1-(4-amino-5-bromopyrimidin-2-yl)-4-methylpiperidin-4-yl)carbamate (880mg, 2.28 mmol). MS m/z 387.3 (M+H)⁺.

Intermediate 3 tert-butyl(3-bromo-6-(4-((tert-butoxycarbonyl)amino)-4-methylpiperidin-1-yl)pyrazin-2-yl)(tert-butoxycarbonyl)carbamate

Step a: A solution of 3-bromo-6-chloropyrazin-2-amine (10.0 g, 48.0mmol), DMAP (2.99 g, 24.47 mmol) and, di-tert-butyl dicarbonate (26.2 g,120 mmol) in DCM (96 mL) was stirred 16 h at RT. The reaction mixturewas diluted with brine and extracted with EtOAc. The combined organicphases were dried over MgSO₄, filtered, concentrated and the resultingresidue was purified by silica chromatography (0 to 30% gradient ofEtOAc/heptane) to give tert-butyl(3-bromo-6-chloropyrazin-2-yl)carbamate (18.77 g, 45.9 mmol). ¹H NMR(400 MHz, CHLOROFORM-d) δ ppm 8.36 (s, 1 H), 1.45 (s, 18 H).

Step b: A solution of tert-butyl (3-bromo-6-chloropyrazin-2-yl)carbamate(11.9 g, 29.1 mmol), tert-butyl (4-methylpiperidin-4-yl)carbamate (12.48g, 58.2 mmol), and 4-methylmorpholine (3.84 mL, 34.9 mmol) in NMP (58mL) was stirred 16 h at 90° C. After cooling to RT, the reaction mixturewas poured into H₂O (300 mL) and the solid formed was filtered off. Thissolid was further purified by silica chromatography (0 to 30% gradientof EtOAc/heptane (containing 0.25% v/v of Et₃N)) to give tert-butyl(3-bromo-6-(4-((tert-butoxycarbonyl)amino)-4-methylpiperidin-1-yl)pyrazin-2-yl)(tert-butoxycarbonyl)carbamate(80% purity). This compound was further purified by silicachromatography (0 to 7% gradient of EtOAc/DCM (containing 0.25% v/v ofEt₃N)) to give tert-butyl(3-bromo-6-(4-((tert-butoxycarbonyl)amino)-4-methylpiperidin-1-yl)pyrazin-2-yl)(tert-butoxycarbonyl)carbamate(7.02 g, 11.97 mmol). MS m/z 586.5 (M+H)⁺.

Intermediate 4 tert-butyl(1-(6-amino-5-bromopyrazin-2-yl)-4-methylpiperidin-4-yl)carbamate

A solution of tert-butyl (3-bromo-6-chloropyrazin-2-yl)carbamate (8.184g, 20.03 mmol), tert-butyl (4-methylpiperidin-4-yl)carbamate (8.58 g,40.1 mmol), and 4-methylmorpholine (2.64 mL, 24.03 mmol) in NMP (50 mL)was stirred 16 h at 150° C. After cooling to RT, the reaction mixturewas diluted with aqueous NaHCO₃ and extracted with Et₂O. The combinedorganic phases were dried over MgSO₄, filtered, concentrated, and theresulting residue was purified by silica chromatography (4 to 40%gradient of EtOAc/heptane) to give tert-butyl(1-(6-amino-5-bromopyrazin-2-yl)-4-methylpiperidin-4-yl)carbamate (1.629g, 4.01 mmol). MS m/z 388.0 (M+H)⁺.

Intermediate 53-bromo-6-((3S,5R)-3,5-dimethylpiperazin-1-yl)pyrazin-2-amine

A suspension of 3-bromo-6-chloropyrazin-2-amine (250 mg, 1.199 mmol) and(2S,6R)-2,6-dimethylpiperazine (151 mg, 1.319 mmol) in DIPEA (1.5 mL,8.59 mmol) was stirred for 16 h at 130° C. After cooling to RT, thevolatiles were removed under reduced pressure, the resulting solid wassuspended in H₂O, filtered and the solid was further purified by HPLC(gradient elution 35-60% acetonitrile in water, 5 mM NH₄OH modifier) togive 3-bromo-6-((3S,5R)-3,5-dimethylpiperazin-1-yl)pyrazin-2-amine (40.0mg, 0.140 mmol). MS m/z (M+H)⁺ 287.0.

Intermediate 66-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine

A suspension of 3-bromo-6-chloropyrazin-2-amine (1.2 g, 5.76 mmol),(2,3-dichlophenyl)boronic acid (1.1 g, 5.76 mmol), potassium phosphate(3.67 g, 17.27 mmol), and PdCl₂(dppf).DCM adduct (235 mg, 0.288 mmol) inMeCN:H₂O (9:1, 15 mL, degassed) was stirred in a microwave reactor for 4h at 120° C. After cooling to RT, the reaction was filtered through apad of Celite followed by EtOAc (25 mL) wash. The combined filtrateswere concentrated and the resulting residue was purified by silicachromatography (0 to 30% gradient of EtOAc/heptane) to give6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine(633 mg, 2.306 mmol). MS m/z 276.4 (M+H)⁺.

Intermediate 7 tert-butyl(1-(5-amino-6-bromo-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate

Step a: A solution of 6-azauracil (1.0 g, 8.84 mmol), POCl₃ (10 mL, 107mmol), and N,N-dimethylaniline (2 mL, 1.784 mmol) was stirred in amicrowave reactor for 25 min at 90° C. After cooling to RT, the reactionwas poured into a beaker containing heptane (200 mL) stirred for 5 minat RT and the phases were separated. This procedure was repeated twice(200 mL of heptane each). The heptane phases were filtered through a padof Celite and MgSO₄, the volatiles were removed under reduced pressureand the resulting residue was treated with NH₃ (7 N in MeOH, 5 mL in 10mL of MeOH) precooled at 0° C. The mixture was stirred for 5 min at RT,then, the volatiles were removed to give 3-chloro-1,2,4-triazin-5-amine(200 mg, 17.3% yield). This compound was used in next step withoutfurther purification.

Step b: A solution of 3-chloro-1,2,4-triazin-5-amine (165 mg, 1.264mmol), tert-butyl (4-methylpiperidin-4-yl)carbamate (271 mg, 1.264mmol), and 4-methylmorpholine (208 μL, 1.896 mmol) in NMP (5 mL) wasstirred in a microwave reactor for 3 h at 130° C. After cooling to RT,the resulting residue was purified by HPLC (gradient elution 15-40%acetonitrile in water, 5 mM NH₄OH modifier) to give tert-butyl(1-(5-amino-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate (71.0mg, 0.23 mmol). MS m/z 308.4 (M+H)⁺.

Step c: A solution of tert-butyl(1-(5-amino-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate (71 mg,0.230 mmol) and NBS (41 mg, 0.230 mmol) in CHCl₃ (2 mL) was stirred 16 hat RT. The volatiles were removed under reduced pressure and theresulting residue was purified by silica chromatography (0 to 5%gradient of MeOH(containing 1% NH₃)/DCM) to give tert-butyl(1-(5-amino-6-bromo-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate(87 mg, 0.23 mmol). MS m/z 387.3 (M+H)⁺.

Intermediate 8 tert-butyl ((4-propylpiperidin-4-yl)methyl)carbamate

Step a: To a solution of N,N-diisopropylamine (14.0 mL, 97.5 mmol) inTHF (100 mL) was added (at −78° C. and under N₂) n-butyllithium (1.6 Min hexane; 59.0 mL, 94.25 mmol) dropwise. The resulting mixture wasstirred for 30 min at RT. 1-benzyl piperidine-4-carbonitrile (6.5 g,32.5 mmol) in THF (50 mL) was added at −78° C. After stirring for 30 minat this temperature, n-propyl iodide (20.5 mL, 211.3 mmol) was added.The resulting mixture was stirred at −78° C. for 1 h. The mixture wasquenched by addition of saturated aqueous ammonium chloride solution andit was extracted with EtOAc. The combined organic phases were washedwith brine, dried over Na₂SO₄, filtered and concentrated to obtain1-benzyl-4-propylpiperidine-4-carbonitrile (6.0 g, 24.8 mmol). Thiscompound was used without further purification. MS m/z 243 (M+H)⁺.

Step b: To a suspension of 1-benzyl-4-propylpiperidine-4-carbonitrile(1.0 g, 4.1 mmol) Boc₂O (2.84 mL, 12.4 mmol), and NiCl₂.6H₂O (0.195 g,0.82 mmol) in MeOH (20 mL) was added at 0° C. sodium borohydride (1.0 g,28.9 mmol) in portions. The resulting mixture was stirred for 12 h atRT. The volatiles were removed under reduced pressure. The resultingresidue was dissolved in DCM, filtered through Celite and the volatileswere removed under reduced pressure. The resulting residue was purifiedby silica chromatography (0 to15% gradient of EtOAc/heptane) to givetert-butyl ((1-benzyl-4-propylpiperidin-4-yl)methyl)carbamate (0.8 g,2.3 mmol). MS m/z 347 (M+H)⁺.

Step c: A suspension of tert-butyl((1-benzyl-4-propylpiperidin-4-yl)methyl)carbamate (5.0 g, 14.4 mmol)and 10% Pd/C (2 g) in MeOH (100 mL) was vigorously stirred under H₂(using a balloon) for 4 h at RT. The mixture was filtered through Celitefollowed by MeOH wash. The volatiles were removed under reduced pressureand the resulting residue was triturated from pentane to give tert-butyl((4-propylpiperidin-4-yl)methyl)carbamate (2.7 g, 10.5 mmol) as a whitepowder. ¹H NMR (400 MHz, DMSO-d₆) δ 6.68 (m, 1 H), 2.87 (d, J=6.4 Hz, 2H), 2.69-2.53 (m, 4 H), 1.38 (s, 9 H), 1.18 (m, 8 H), 0.84 (q, J=5.9 Hz,3 H). MS m/z 257 (M+H)⁺.

Intermediate 92-((4-(pyridin-3-yl)piperidin-4-yl)methyl)isoindoline-1,3-dione

Step a: To a suspension of sodium hydride (60% in mineral oil, 1.487 gmg, 37.2 mmol) in DMF (25 mL) was added at 0° C.2-(pyridin-3-yl)acetonitrile (1.537 g, 13.01 mmol) in DMF (5 mL)dropwise within 10 min The resulting mixture was stirred 30 min at 0° C.Tert-butyl-bis(2-chloroethyl)carbamate (3.0 g, 12.39 mmol) in DMF (5 mL)was added at 0° C., the resulting mixture was stirred for 15 min at 0°C. and for 16 h at 75° C. After cooling to RT, the reaction mixture wasdiluted with NaHCO₃ aq. and extracted with Et₂O. The combined organicphases were dried over MgSO₄, filtered, concentrated and the resultingresidue was purified by silica chromatography (10 to 80% gradient ofEtOAc/heptane) to give tert-butyl4-cyano-4-(pyridin-3-yl)piperidine-1-carboxylate (2.58 g, 8.98 mmol). ¹HNMR (400 MHz, CHLOROFORM-d) δ ppm 8.69 (d, J=2.02 Hz, 1 H), 8.55 (dd,J=4.80, 1.52 Hz, 1 H), 7.68-7.81 (m, 1 H), 7.30 (ddd, J=8.08, 4.80, 0.76Hz, 1 H), 4.25 (br. s., 2 H), 3.15 (br. s., 2 H), 2.06 (d, J=11.87 Hz, 2H), 1.81-1.98 (m, 2 H), 1.42 (s, 9 H).

Step b: To a solution of tert-butyl4-cyano-4-(pyridin-3-yl)piperidine-1-carboxylate (1.0 g, 3.48 mmol) inMeOH (30 mL) was added at RT, CoCl₂.6H₂O (828 mg, 2.38 mmol) and sodiumborohydride (658 mg, 17.40 mmol). The resulting mixture was stirred for1 h at RT. The volatiles were removed under reduced pressure and theresulting residue was purified by silica chromatography (0 to 15%gradient of MeOH/DCM) to give tert-butyl4-(aminomethyl)-4-(pyridin-3-yl)piperidine-1-carboxylate (683 mg, 2.344mmol). MS m/z 292.2 (M+H)⁺.

Step c: A suspension of tert-butyl4-(aminomethyl)-4-(pyridin-3-yl)piperidine-1-carboxylate (683 mg, 2.344mmol), phthalic anhydride (434 mg, 2.93 mmol), activated molecularsieves (3 angstroms, 500 mg), and DIPEA (1.23 mL, 7.03 mmol) in toluene(12 mL) was stirred for 16 h at 105° C. After cooling to RT, the mixturewas filtered through a pad of Celite followed by EtOAc (10 mL) wash. Thevolatiles were removed under reduced pressure and the resulting residuewas purified by silica chromatography (0 to 10% gradient of MeOH/DCM) togive tert-butyl4-((1,3-dioxoisoindolin-2-yl)methyl)-4-(pyridin-3-yl)piperidine-1-carboxylate(640 mg, 1.518 mmol). MS m/z 422.3 (M+H)⁺.

Step d: A solution of tert-butyl4-((1,3-dioxoisoindolin-2-yl)methyl)-4-(pyridin-3-yl)piperidine-1-carboxylate(640 mg, 1.518 mmol) and HCl (4M in dioxane, 1.9 mL, 7.59 mmol) indioxane (20 mL) was stirred for 16 h at RT. The volatiles were removedunder reduced pressure to give the HCl salt of the title compound (543mg, 1.518 mmol). MS m/z 322.2 (M+H)⁺.

Intermediate 10 tert-butyl((4-(pyrazin-2-yl)piperidin-4-yl)methyl)carbamate

Step a: To a suspension of sodium hydride (60% in mineral oil, 1.90 g,47.7 mmol) in DMF (30 mL) was added at 0° C.2-(pyrazin-2-yl)acetonitrile (1.90 g, 15.90 mmol) in DMF (5 mL) dropwisewithin 10 min. The resulting mixture was stirred 30 min at 0° C.N-benzyl-2-chloro-N-(2-chloroethyl)ethanamine (4.7 g, 17.5 mmol) in DMF(5 mL) was added at 0° C., the resulting mixture was stirred for 15 minat 0° C. and for 16 h at 90° C. After cooling to RT, the reactionmixture was diluted with NaHCO₃ aq. and extracted with EtOAc (3×25 mL).The combined organic phases were dried over Na₂SO₄, filtered,concentrated, and the resulting residue was purified by trituration withhexane to give 1-benzyl-4-(pyrazin-2-yl)piperidine-4-carbonitrile (1.60g, 5.76 mmol).

Step b: To a solution of1-benzyl-4-(pyrazin-2-yl)piperidine-4-carbonitrile (1.50 g, 5.39 mmol)in NH₃ (7 N in MeOH, 50 mL) was added at RT Raney nickel (750 mg). Theresulting suspension was vigorously stirred under H₂ (60 psi) at RTuntil the starting material disappeared (16 h). The reaction mixture wasfiltered through a pad of Celite followed by MeOH (50 mL) wash. Thevolatiles were removed under reduced pressure to give(1-benzyl-4-(pyrazin-2-yl)piperidin-4-yl)methanamine (1.20 g, 4.25 mmol)which was used in next step without further purification. MS m/z 319(M+H)⁺.

Step c: A solution of(1-benzyl-4-(pyrazin-2-yl)piperidin-4-yl)methanamine (1.20 g, 4.25mmol), Et₃N (1.17 mL, 8.51 mmol), and Boc₂O (1.95 mL, 8.51 mmol) in DCM(50 mL) was stirred for 2 h at RT. The reaction was diluted with H₂O andit was extracted with DCM (3×25 mL). The combined organic phases werewashed with brine, dried over Na₂SO₄, filtered and the volatiles wereremoved under reduced pressure. The resulting residue was purified bysilica chromatography (0 to 100% gradient of EtOAc/heptane) to givetert-butyl ((1-benzyl-4-(pyrazin-2-yl)piperidin-4-yl)methyl)carbamate(1.30 g, 3.40 mmol). MS m/z 383 (M+H)⁺.

Step d: A suspension of tert-butyl((1-benzyl-4-(pyrazin-2-yl)piperidin-4-yl)methyl)carbamate (1.50 g, 3.93mmol) and Pd(OH)₂ (20% on carbon, 600 mg, 50% moisture) in MeOH (20 mL)was vigorously stirred under H₂ (50 psi) for 3 h at RT. The reactionmixture was filtered through a pad of Celite followed by MeOH (50 mL)wash. The volatiles were removed under reduced pressure and to givetert-butyl ((4-(pyrazin-2-yl)piperidin-4-yl)methyl)carbamate (1.10 g,3.76 mmol) which was used without further purification. MS m/z 283(M+H)⁺.

The following intermediates were made using the above procedure ormodifications to the above procedure using the corresponding commercialavailable heteroaromatic nitriles.

TABLE 1 Intermediate Structure 11

12

Intermediate 13 racemic trans-4-amino-4-methylpiperidin-3-ol

Step a: Commercially available1-benzyl-4-methyl-1,2,3,6-tetrahydropyridine was converted to racemic3-benzyl-6-methyl-7-oxa-3-azabicyclo[4.1.0]heptane by procedure a, asdescribed in Grishina et al., Russian Journal of Organic Chemistry, vol.41, No. 2, 2005.

Step b: To a 0° C. solution of racemic3-benzyl-6-methyl-7-oxa-3-azabicyclo[4.1.0]heptane (4.5 g, 22.153 mmol)in water (45 mL) and acetic acid (22 mL) was added sodium azide (7.2 g,110.766 mmol). The reaction was warmed to RT and stirred for 16 h. Thereaction was quenched by addition of saturated aq sodium bicarbonate (60mL). The mixture was extracted with DCM (3×200 mL) and EtOAc (2×150 mL)and the combined organic extracts were washed with brine, dried overNa₂SO₄, filtered, and concentrated. The crude product was purified bysilica gel chromatography (25 to 30% gradient of EtOAc/heptane) to giveracemic trans-4-azido-1-benzyl-4-methylpiperidin-3-ol (3.3 g, 16.23mmol). ¹H NMR (400 MHz, CDCl₃): δ ppm 7.34-7.24 (m, 5 H), 3.53 (s, 2 H),3.35 (s, 1 H), 2.70-2.57 (m, 3 H), 3.32-2.26 (m, 1 H), 1.89-1.81 (m, 1H), 1.59-1.55 (m, 1 H), 1.37 (s, 3 H). MS m/z 247.4 (M+H)⁺.

Step c: To a solution of racemictrans-4-azido-1-benzyl-4-methylpiperidin-3-ol (474 mg, 1.926 mmol) inMeOH was added a catalytic amount of Pd/C. The solution was degassed andbackfilled with H₂ 2×. The reaction was stirred for 1 h, LCMS indicatedthat SM was consumed. The reaction was filtered through Celite/sand(MeOH). This resulted in the isolation of 444 mg (yellow oil/semisolid).¹H NMR indicated incomplete reduction (aromatic peaks detected), so theabove procedure was repeated. The reaction was filtered throughCelite/sand and concentrated. ¹H NMR confirmed the absence of aromaticpeaks. 247 mg of a viscous oil resulted which was used with no furtherpurification.

Intermediate 14 racemic cis-4-azido-4-methylpiperidin-3-yl benzoate

Step a: To a solution of racemictrans-4-azido-1-benzyl-4-methylpiperidin-3-ol (500 mg, 2.03 mmol) in DCM(20 mL) was added benzoic acid (273 mg, 2.233 mmol),di-tert-butylazodicarboxylate (514 mg, 2.233 mmol), andtriphenylphosphine (586 mg, 2.233 mmol). The reaction was stirred for 16h at RT. The reaction was diluted with DCM (100 mL) and washed withsaturated aq sodium bicarbonate (100 mL). The organic layer was driedover MgSO₄, filtered, and concentrated and the resulting residue waspurified by silica chromatography (0 to 20% gradient of EtOAc/heptane)to give racemic cis-4-azido-1-benzyl-4-methylpiperidin-3-yl benzoate (92mg, 0.263 mmol) as a yellow oil which solidified to an off-whitecrystalline solid. ¹H NMR (DMSO-d₆) δ ppm 8.37 (d, J=9.0 Hz, 2 H), 8.19(d, J=8.9 Hz, 2 H), 7.31-7.18 (m, 6 H), 4.44-4.32 (m, 2 H), 4.08 (d,J=13.3 Hz, 1 H), 3.60 (d, J=13.3 Hz, 1 H), 3.0 (dd, J=4.4, 6.4 Hz, 1 H),2.79-2.73 (m, 1 H), 2.49-2.42 (m, 1 H), 1.92-1.85 (m, 2 H), 1.49 (s, 3H). MS m/z 351.1 (M+H)⁺.

Step b: To a solution of racemiccis-4-azido-1-benzyl-4-methylpiperidin-3-yl benzoate (90 mg, 0.257 mmol)in MeOH (50 mL) was added 10% Pd/C (50 mg). The reaction was degassedand backfilled 3× with H₂ (balloon). The reaction was stirred for 16 h.The crude reaction mixture was filtered through Celite (MeOH) and thesolution collected was concentrated, to give racemiccis-4-azido-4-methylpiperidin-3-yl 4-nitrobenzoate (48 mg, 0.184 mmol).The crude material was used without further purification. MS m/z 235.2(M+H).

Intermediate 15 racemic tert-butyltrans-((3-hydroxy-4-methylpiperidin-4-yl)methyl)carbamate

Step a: A solution of lithium hydride (0.118 g, 14.8 mmol) in THF (20mL) was added at 0° C. acetone cyanohydrin (1.4 mL, 14.8 mmol). Theresulting reaction mixture was stirred for 2 h at RT. The volatiles wereremoved under reduced pressure to give a white solid. To a solution ofthis solid in THF (60 mL) was added at RT3-benzyl-6-methyl-7-oxa-3-azabicyclo[4.1.0]heptane (2.0 g, 9.85 mmol)dropwise. After complete addition, the solution was stirred for 14 hunder reflux. After cooling to RT, water (10 mL) was added and theresulting mixture was extracted with EtOAc (3×100 mL). The combinedorganic phases were washed with brine, dried over Na₂SO₄, filtered andconcentrated. The resulting residue was purified by silicachromatography (0 to 20% gradient of EtOAc/heptane) to obtain racemictrans-1-benzyl-3-hydroxy-4-methylpiperidine-4-carbonitrile (0.70 g, 3.0mmol). ¹H NMR (400 MHz, DMSO-d₆) δ 7.36-7.22 (m, 5 H), 5.25 (d, J=6.0Hz, 1 H), 3.70-3.67 (m, 1 H), 3.49 (dd, J=13.2, 10.4 Hz, 2 H), 2.37 (m,3 H), 1.88-1.74 (m, 2 H), 1.25 (s, 3 H). MS m/z 231.2 (M+H)⁺.

Step b: A suspension of racemic trans-1-benzyl-3-hydroxy-4-methylpiperidine-4-carbonitrile (1.3 g, 5.6 mmol) and Raney nickel (50% inwater, 600 mg) in ammonia (7 N in EtOH; 80 mL) was vigorously stirredunder hydrogen (balloon) for 6 h at RT. The mixture was filtered throughCelite under N₂ and washed with MeOH. The volatiles were removed underreduced pressure to givetrans-4-(aminomethyl)-1-benzyl-4-methylpiperidin-3-ol (1.6 g, 4.79mmol). This compound was used in next step without further purification.MS m/z 235.2 (M+H)⁺.

Step c: A solution oftrans-4-(aminomethyl)-1-benzyl-4-methylpiperidin-3-ol (1.6 g, 4.79mmol), Boc₂O (2.84 mL, 12.4 mmol), and NaHCO₃ (0.935 g, 11.1 mmol) inCHCl₃ (70 mL) was stirred for 14 h at RT. The mixture was diluted withDCM and washed with ice water, brine, dried over Na₂SO₄, filtered andconcentrated. The resulting residue was purified by silicachromatography (0 to 5% gradient of MeOH/DCM) to give racemic tert-butyltrans-(1-benzyl-3-hydroxy-4-methylpiperidin-4-yl)methyl)carbamate (1.6g, 4.79 mmol). MS m/z 335.3 (M+H)⁺.

Step d: A suspension of racemic tert-butyltrans-((1-benzyl-3-hydroxy-4-methylpiperidin-4-yl)methyl)carbamate (1.1g, 3.3 mmol) and Pd(OH)₂ (20% on charcoal; 0.250 g) in MeOH (60 mL) wasvigorously stirred under hydrogen atmosphere (balloon) for 6 h at RT.The resulting mixture was filtered through Celite and washed with MeOH.Concentrated, then triturated from hexane (10 mL) and diethyl ether (2mL) to give racemic tert-butyltrans-((3-hydroxy-4-methylpiperidin-4-yl)methyl)carbamate (0.70 g, 2.87mmol) as a white powder. ¹H NMR (400 MHz, METHANOL-d₄) δ 3.42 (dd,J=9.9, 4.4 Hz, 1 H), 3.12 (d, J=13.9 Hz, 1 H), 2.94-2.84 (m, 2 H),2.82-2.68 (m, 2 H), 2.62 (dd, J=12.5, 10.0 Hz, 1 H), 1.44 (s, 9 H),1.41-1.30 (m, 2 H), 0.91 (s, 3 H). MS m/z 245.1 (M+H)⁺.

Intermediate 16 racemic tert-butylcis-((3-hydroxy-4-methylpiperidin-4-yl)methyl)carbamate

Step a: A solution of racemic trans-1-benzyl-3-hydroxy-4-methylpiperidine-4-carbonitrile (2.0 g, 8.70 mmol), triphenylphosphine (3.41g, 13.0 mmol), and diisopropylazodicarboxylate (2.63 g, 13.0 mmol) inTHF (30 mL) was stirred for 10 min at 0° C. 4-Nitrobenzoic acid (2.18 g,13.0 mmol) was added portionwise and the resulting mixture was stirredfor 16 h at RT. The mixture was diluted with water and extracted withEtOAc. The combined organic phases were washed with brine, dried overNa₂SO₄, filtered and concentrated. The resulting residue was trituratedwith MeOH to give racemiccis-1-benzyl-4-cyano-4-methylpiperidin-3-yl4-nitrobenzoate (1.5 g, 3.96mmol). MS m/z 380 (M+H)⁺. This material was used without furtherpurification.

Step b: A solution of racemiccis-1-benzyl-4-cyano-4-methylpiperidin-3-yl 4-nitrobenzoate (1.5 g, 3.96mmol) and potassium carbonate (1.07 g, 7.92 mmol) in MeOH (20 mL) wasvigorously stirred for 10 min at 0° C. and for 1 h at RT. The volatileswere removed under reduced pressure. The resulting residue was dilutedwith water and extracted with EtOAc (3×). The combined organic phaseswere washed with brine, dried over Na₂SO₄, filtered and concentrated.The resulting residue was purified by silica chromatography (0 to 15%gradient of EtOAc/heptane) to give racemiccis-1-benzyl-3-hydroxy-4-methylpiperidine-4-carbonitrile (0.8 g, 3.5mmol). ¹H NMR (400 MHz, CDCl₃) δ 7.36-7.26 (m, 5 H), 3.99 (d, J=12.4 Hz,1 H), 3.67 (d, J=12.8, 1 H), 3.60-3.51 (m, 2 H), 3.11-3.07 (m, 2 H),2.76-2.69 (m, 2 H), 2.24 (dd, J=12.8, 6.0 Hz, 1 H), 1.87-1.80 (m, 1 H),1.54 (s, 3 H). MS m/z 231 (M+H)⁺.

Step c: A suspension ofcis-1-benzyl-3-hydroxy-4-methylpiperidine-4-carbonitrile (800 mg, 3.5mmol) and Raney nickel (50% in water, 700 mg) in ammonia (7 N in EtOH;20 mL) was vigorously stirred under hydrogen (balloon) for 16 h at RT.The mixture was filtered through Celite under N₂ and washed with MeOH.The volatiles were removed under reduced pressure to give racemiccis-4-(aminomethyl)-1-benzyl-4-methylpiperidin-3-ol (700 mg, 3.0 mmol).This compound was used in next step without further purification. MS m/z235.2 (M+H)⁺.

Step d: A solution ofcis-4-(aminomethyl)-1-benzyl-4-methylpiperidin-3-ol (700 mg, 3.0 mmol),Boc₂O (1.1 mL, 2.99 mmol), and Et₃N (860 μL, 5.98 mmol) in DCM (10 mL)was stirred for 2 h at RT. The mixture was diluted with DCM and washedwith ice water, brine, dried over Na₂SO₄, filtered and concentrated. Theresulting residue was purified by silica chromatography (0 to 50%gradient of EtOAc/heptane) to give racemic tert-butylcis-(1-benzyl-3-hydroxy-4-methylpiperidin-4-yl)methyl)carbamate (700 mg,2.10 mmol). This compound was used in next step without furtherpurification. MS m/z 335 (M+H)⁺.

Step e: A suspension of racemic tert-butylcis-(1-benzyl-3-hydroxy-4-methylpiperidin-4-yl)methyl)carbamate (700 mg,2.1 mmol) and Pd/C (10% on charcoal; 300 mg) in MeOH (20 mL) wasvigorously stirred under hydrogen atmosphere (balloon) for 5 h at RT.The resulting mixture was filtered through Celite and washed with MeOHand concentrated. The resulting residue was purified by silicachromatography (0 to 10% gradient of MeOH/DCM) to give racemictert-butyl cis-((3-hydroxy-4-methylpiperidin-4-yl)methyl)carbamate (200mg, 0.8 mmol) as a white powder. ¹H NMR (400 MHz, METHANOL-d₄) δ3.73-3.67 (m, 1 H), 3.59 (dd, J=11.1, 7.7 Hz, 1 H), 3.15-2.99 (m, 4 H),1.90 (m, 1 H), 1.62 (m, 1 H), 1.47 (m, 1 H), 1.44 (s, 9 H), 0.96 (s, 3H). MS m/z 245 (M+H)⁺.

Example 12-(4-amino-4-methylpiperidin-1-yl)-5-(2,3-dichlorophenyl)pyrimidin-4-amine

Step a: A suspension of tert-butyl(1-(4-amino-5-bromopyrimidin-2-yl)-4-methylpiperidin-4-yl)carbamate (966mg, 2.50 mmol), (2,3-dichlophenyl)boronic acid (596 mg, 3.13 mmol),potassium phosphate (1.59 g, 7.50 mmol), and PdCl₂(dppf).DCM adduct (204mg, 0.25 mmol) in MeCN:H₂O (9:1, 10 mL, degassed) was stirred in amicrowave reactor for 3 h at 110° C. After cooling to RT, the reactionwas filtered through a pad of Celite followed by EtOAc (50 mL) wash. Thecombined filtrates were concentrated and the resulting residue waspurified by silica chromatography (0 to 5% gradient of MeOH/DCM) to givetert-butyl(1-(4-amino-5-(2,3-dichlorophenyl)pyrimidin-2-yl)-4-methylpiperidin-4-yl)carbamate(1.07 g, 2.37 mmol). MS m/z 452.4 (M+H)⁺.

Step b: To a solution of tert-butyl(1-(4-amino-5-(2,3-dichlorophenyl)pyrimidin-2-yl)-4-methylpiperidin-4-yl)carbamate(2.14 g, 4.73 mmol) in DCM (50 mL), was added TFA (31.7 mL). Afterstirring at 0° C. until no starting material remained (30 min, monitoredby LCMS), the volatiles were removed under reduced pressure and theresulting residue was purified by HPLC (gradient elution 10-30%acetonitrile in water, 0.1% TFA modifier), to give the title compound(657 mg, 1.69 mmol; HCl salt). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm7.49-7.63 (m, 2 H), 7.34 (t, J=7.71 Hz, 1 H), 7.26 (d, J=7.33 Hz, 1 H),4.18 (d, J=12.13 Hz, 2 H), 3.38-3.61 (m, 2 H), 1.88 (br. s., 4 H), 1.45(s, 3 H). HRMS calcd for C₁₆H₂₀Cl₂N₅ (M+H)⁺ 352.1096, found 352.1086.IC₅₀ is 0.258 μM.

The following compounds were made using the above procedure ormodifications to the above procedure using the corresponding boronicacid.

TABLE 2 Example Compound Characterization IC₅₀ (μM) 2

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.50 (s, 1 H), 7.19-7.25 (m, 1 H),7.12-7.19 (m, 1 H), 6.98-7.04 (m, 1 H), 3.88 (ddd, J = 13.64, 6.32, 4.55Hz, 2 H), 3.48-3.56 (m, 2 H), 2.33 (s, 3 H), 1.47- 1.54 (m, 4 H), 1.16(s, 3 H). HRMS calcd for C₁₇H₂₂ClN₅ (M + H)⁺ 332.1642, found 332.1645.0.858 3

as TFA salt. ¹H NMR (400 MHz, DMSO- d₆) δ ppm 7.92 (br. s., 4 H), 7.88(s, 1 H), 7.16 (d, J = 3.76 Hz, 1 H), 6.99 (d, J = 3.76 Hz, 1 H),4.14-4.24 (m, 2 H), (piperidine methylene hidden by H₂O peak, 2 H), 1.68(br. s., 4 H), 1.36 (s, 3 H). HRMS calcd for C₁₄H₁₈ClN₅S (M + H)⁺324.1050, found 324.1045 0.950 4

as TFA salt. ¹H NMR (400 MHz, DMSO- d₆) δ ppm 7.85-8.00 (m, 4 H), 7.83(s, 1 H), 6.94 (br. s., 1 H), 6.84 (br. s., 1 H), 4.11-4.23 (m, 2 H),(piperidine methylene hidden by H₂O peak, 2 H), 2.46 (s, 3 H), 1.69 (br.s., 4 H), 1.37 (s, 3 H). HRMS calcd for C₁₅H₂₁N₅S (M + H)⁺ 304.1596,found 304.1591 0.974 5

as TFA salt. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.70 (s, 1 H), 7.27 (d,J = 3.03 Hz, 1 H), 6.96 (d, J = 2.78 Hz, 1 H), 4.27 (d, J = 13.64 Hz, 2H), 3.86 (s, 3 H), 3.48-3.69 (m, 2 H), 1.98 (d, J = 5.05 Hz, 4 H), 1.55(s, 3 H). HRMS calcd for C₁₇H₂₂Cl₂N₅O (M + H)⁺ 382.1201, found 382.12562.859

Example 63-(4-amino-2-(4-amino-4-methylpiperidin-1-yl)pyrimidin-5-yl)-4,5-dichlorophenol

To a solution2-(4-amino-4-methylpiperidin-1-yl)-5-(2,3-dichloro-5-methoxyphenyl)pyrimidin-4-amine(77 mg, 0.155 mmol) in DCM (5 mL) was added at −78° C. and under N₂,BBr₃ (1 M in DCM, 465 μL, 0.465 mmol). After stirring 16 h at RT, thevolatiles were removed under reduced pressure and the resulting residuewas purified by HPLC (gradient elution 10-30% acetonitrile in water, 5mM NH₄OH modifier) to give the title compound (29.2 mg, 0.079 mmol). ¹HNMR (400 MHz, METHANOL-d₄) δ ppm 7.51 (s, 1 H), 6.83 (d, J=3.01 Hz, 1H), 6.53 (d, J=2.76 Hz, 1 H), 3.84-4.02 (m, 2 H), 3.40-3.62 (m, 2 H),1.40-1.63 (m, 4 H), 1.19 (s, 3 H). HRMS calcd for C₁₆H₂₀Cl₂N₅O (M+H)⁺368.1104, found 368.1086. IC₅₀ is 0.225 μM.

Example 76-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine

A solution of6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine(950 mg, 3.46 mmol), tert-butyl (4-methylpiperidin-4-yl)carbamate (1.48g, 6.92 mmol) and, N-methylmorphpoline (761 μL, 6.92 mmol) in NMP (9 mL)was was stirred in a microwave reactor for 2 h at 250° C. Note thatunder these reaction conditions, the Boc protecting group was removed.After cooling to RT, the reaction was purified by HPLC (gradient elution15-40% acetonitrile in water, 0.1% TFA modifier) to give the titlecompound (750 mg, 01.885 mmol, HCl salt). ¹H NMR (400 MHz, METHANOL-d₄)δ ppm 7.81 (dd, J=6.69, 2.91 Hz, 1 H), 7.62 (s, 1 H), 7.45-7.59 (m, 2H), 4.25 (br. s., 2 H), 3.44-3.61 (m, 2 H), 1.81-2.04 (m, 4 H), 1.55 (s,3 H). HRMS calcd for C₁₆H₂₀Cl₂N (M+H)⁺ 352.1096, found 352.1099. IC₅₀ is0.071 μM.

The following compounds were made using the above procedure ormodifications to the above procedure using the corresponding amine.Although in most cases the Boc protecting group was removed under thereaction conditions, HCl or TFA was used when necessary.

TABLE 3 Example Compound Characterization IC₅₀ (μM) 8

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.70-7.68 (m, 1 H), 7.49-7.41 (m, 3H), 4.01 (dt, J₁ = 13.7, J₂ = 4.3, 4.3 Hz, 2 H), 3.48-3.41 (m, 2 H),2.91 (s, 2 H), 1.66- 1.55 (m, 4 H), 1.19 (s, 3H). HRMS calcd forC₁₇H₂₂Cl₂N₅ (M + H)⁺ 366.1252, found 366.1240. 0.076 9

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.62 (dd, J = 8.08, 1.52 Hz, 1 H), 7.48(s, 1 H), 7.41-7.37 (m, 1 H), 7.32-7.29 (m, 1 H), 5.63 (br. S., 2 H),4.31 (d, J = 12.5 Hz, 2 H), 2.86-2.67 (m, 4 H), 2.44 (d, J = 6.3, 2 H),1.78-1.68 (m, 3 H), 1.13-1.03 (m, 2 H). HRMS calcd for C₁₆H₂₀Cl₂N₅ (M +H)⁺ 352.1096, found 352.1087. 0.358 10

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.69-7.66 (m, 1 H), 7.47-7.37 (m, 3H), 4.04 (dt, J₁ = 13.6, J₂ = 4.9, 4.9 Hz, 1 H), 3.95-3.90 (m, 1H),3.79-3.75 (m, 1 H), 3.72-3.66 (m, 1 H), 3.47-3.32 (overlapping withmethanol, m, 3 H), 3.09-3.04 (m, 1 H), 2.69-2.63 (m, 1 H), 2.50-2.45 (m,1 H), 2.25-2.11 (m, 3 H), 1.97-1.88 (m, 2 H). HRMS calcd for C₁₇H₂₀Cl₂N₅(M + H)⁺ 364.1096, found 364.1083. 0.908 11

Chiral SFC purification performed as follows; column: AD-H 21 × 250 mm,flow rate: 75 g per minute, mobile phase: 40% MeOH and 10 mM NH₄OH inCO₂, detection: 354 mn UV to obtain single enantiomer R_(t) (P1) = 1.9min, R_(t) (P2) = 3.2 min. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.60 (dd, J =7.96, 1.64 Hz, 1 H), 7.35- 7.41 (m, 1 H), 7.30 (dd, J = 7.58, 1.77 Hz, 1H), 7.22 (s, 1 H), 5.49 (s, 2 H), 3.71-3.80 (m, 2 H), 3.37-3.52 (m, 3H), 2.32 (s, 2 H), 1.76-1.93 (m, 2 H), 1.04-1.11 (m, 3 H). HRMS calcdfor C₁₇H₂₂Cl₂N₅O (M + H)⁺ 382.1201, found 382.1220. P1 = 0.038 P2 =0.164 12

¹H NMR (400 MHz, METHANOL-d₄) δ 7.80 (dd, J = 2.27, 7.33 Hz, 1 H),7.48-7.62 (m, 7 H), 7.37-7.46 (m, 1 H), 4.22 (br. s., 2 H), 3.37 (m, 2H), 3.22 (s, 2 H), 2.54 (d, J = 14.15 Hz, 2 H), 1.99 (ddd, J = 3.66,10.74, 14.15 Hz, 2 H). HRMS calcd for C₂₂H₂₄C1₂N₅ (M + H)⁺ 428.1409,found 428.1385. 0.065 13

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.80 (dd, J = 2.01, 7.53 Hz, 1 H),7.46- 7.57 (m, 2 H), 7.29 (s, 1 H), 3.97 (d, J = 11.04 Hz, 2 H),3.65-3.75 (m, 2 H), 2.55 (s, 1 H), 2.26 (br. s., 2 H). HRMS calcd forC₁₅H₁₆Cl₂N₅ (M + H)⁺ 336.0782, found 336.0808. 0.065 14

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.80 (dd, J = 2.38, 7.15 Hz, 1 H),7.48- 7.56 (m, 2 H), 7.28 (s, 1 H), 3.83-3.93 (m, 1 H), 3.77 (d, J =7.03 Hz, 1 H), 3.55-3.65 (m, 1 H), 3.35-3.40 (m, 1 H), 3.06-3.21 (m, 2H), 2.72 (td, J = 7.53, 15.06 Hz, 1 H), 2.32-2.42 (m, 1 H), 1.93 (qd, J= 8.45, 12.55 Hz, 1 H). HRMS calcd for C₁₅H₁₈Cl₂N₅ (M + H)⁺ 338.0939,found 338.0938. 0.068 15

¹H NMR (400 MHz, DMSO-d₆) δ 7.61 (dd, J = 8.0, 1.6 Hz, 1 H), 7.46 (s, 1H), 7.39 (t, J = 7.8 Hz, 1 H), 7.30 (dd, J = 7.7, 1.7 Hz, 1 H), 5.58 (s,2 H), 3.55 (m, 2 H), 3.44 (m, 2 H), 3.32 (m, 2 H), 1.45 (m, 2 H),1.40-1.28 (m, 4 H), 1.27-1.16 (m, 3 H), 0.89 (t, J = 7.0 Hz, 3 H). HRMScalcd for C₁₉H₂₆Cl₂N₅ (M + H)⁺ 394.1565, found 394.1547. 0.075 16

TFA salt. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.66 (dd, J = 1.6, 7.9 Hz, 1H), 7.51 (s, 1 H), 7.43 (t, J = 7.8 Hz, 1 H), 7.36 (dd, J = 1.6, 7.8 Hz,1 H), 4.45-4.31 (m, 2 H), 3.70 (dd, J = 5.0, 10.8 Hz, 1 H), 3.16-3.09(m, 1 H), 2.93 (dd, J = 13.1, 13.1 Hz, 1 H), 1.97-1.91 (m, 1 H),1.89-1.81 (m, 1 H), 1.45 (s, 3 H). HRMS calcd for C₁₆H₂₀N₅OCl₂ (M + H)⁺368.1045, found 368.1049. 0.093 17

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.97 (d, J = 7.1 Hz, 2 H), 8.35 (d,J = 7.1 Hz, 2 H), 7.78 (dd, J = 2.4, 7.2 Hz, 1 H), 7.58 (s, 1 H),7.55-7.45 (m, 2 H), 4.16 (d, J = 13.6 Hz, 2 H), 3.55-3.41 (m, 4 H), 2.56(d, J = 15.2 Hz, 2 H), 2.21 (ddd, J = 3.7, 9.8, 14.1 Hz, 2 H). HRMScalcd for C₂₁H₂₃Cl₂N₆ (M + H)⁺ 429.1361, found 429.1338. 0.102 18

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.94 (d, J = 1.3 Hz, 1 H), 8.77 (dd,J = 1.5, 2.5 Hz, 1 H), 8.65 (d, J = 2.3 Hz, 1 H), 7.81 (dd, J = 3.0, 6.6Hz, 1 H), 7.59 (s, 1 H), 7.54 (d, J = 2.3 Hz, 1 H), 7.52 (s, 1 H),4.22-4.09 (m, 2 H), 3.53-3.41 (m, 4 H), 2.71-2.62 (m, 2 H), 2.04 (ddd, J= 3.8, 9.7, 13.8 Hz, 2 H). HRMS calcd for C₂₀H₂₂Cl₂N₇ (M + H)⁺ 430.1314,found 430.1325. 0.093 19

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.80 (dd, J = 5.15, 0.88 Hz, 1 H)8.22 (t, J = 7.78 Hz, 1 H) 7.91 (d, J = 8.28 Hz, 1 H) 7.82 (dd, J =6.53, 3.01 Hz, 1 H) 7.66 (dd, J = 7.53, 5.27 Hz, 1 H) 7.60 (s, 1 H)7.50-7.58 (m, 2 H) 4.13 (br. s., 2 H) 3.53 (dd, J = 13.18, 9.66 Hz, 2 H)3.45 (s, 2 H) 2.62 (d, J = 15.06 Hz, 2 H) 2.01-2.18 (m, 2 H). HRMS calcdfor C₂₁H₂₃Cl₂N₆ (M + H)⁺ 429.1361, found 429.1357. 0.110 20

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.64 (dd, J = 8.03, 1.51 Hz, 1 H)7.42 (t, J = 7.91 Hz, 1 H) 7.32 (dd, J = 7.65, 1.63 Hz, 1 H) 7.16 (s, 1H) 5.12 (dd, J = 6.40, 3.64 Hz, 1 H) 4.23 (t, J = 8.16 Hz, 1 H)3.77-3.87 (m, 2 H) 3.74 (d, J = 12.30 Hz, 1 H) 3.48 (dd, J = 7.15, 2.89Hz, 1 H) 3.35- 3.41 (m, 1 H) 3.23 (dd, J = 12.42, 3.64 Hz, 1 H). HRMScalcd for C₁₅H₁₆Cl₂N₅ (M + H)⁺ 336.0783, found 336.0780. 0.404 21

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.80 (dd, J = 7.20, 2.40 Hz, 1 H)7.48- 7.57 (m, 2 H) 7.33 (s, 1 H) 3.77-3.89 (m, 2H) 3.60-3.75 (m, 4 H)3.29 (overlapping with methanol, d, 4 H). HRMS calcd for C₁₆H₁₈Cl₂N₅(M + H)⁺ 350.0939, found 350.0913. 0.102

Example 226-(4-amino-4-methylpiperidin-1-yl)-5-bromo-3-(2,3-dichlorophenyl)pyrazin-2-amine

To a solution of6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine(275 mg, 0.590 mmol) in CH₃Cl (7 mL) was added at 0° C. and under N₂,Br₂ (32 μL, 0.619 mmol). After stirring for 2 h at 0° C., the volatileswere removed under reduced pressure and the resulting residue waspurified by HPLC (gradient elution 35-60% acetonitrile in water, 5 mMNH₄OH modifier) to give the title compound (128.8 mg, 0.298 mmol). ¹HNMR (400 MHz, METHANOL-d₄) δ ppm 7.63 (dd, J=7.96, 1.64 Hz, 1 H), 7.42(t, J=7.83 Hz, 1 H), 7.36 (dd, J=7.58, 1.77 Hz, 1 H), 3.52-3.62 (m, 2H), 3.35-3.40 (m, 2 H), 1.76 (t, J=5.56 Hz, 4 H), 1.28 (s, 3 H). HRMScalcd for C₁₆H₁₉BrCl₂N₅ (M+H)⁺ 432.0155, found 432.0157. IC₅₀ is 0.169μM.

Example 236-(4-amino-4-methylpiperidin-1-yl)-5-chloro-3-(2,3-dichlorophenyl)pyrazin-2-amine

To a solution of6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine(HCl salt) (15 mg, 0.043 mmol) in CH₃Cl (250 μL) was added at 0° C. andunder N₂, NBS (7.58 mg, 0.043 mmol) in CH₃Cl (250 μL). After stirringfor 2 h at RT the volatiles were removed under reduced pressure and theresulting residue was purified by HPLC (gradient elution 15-40%acetonitrile in water, 0.1% TFA modifier) to give the title compound(1.7 mg, 3.23 μma TFA salt). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.53(dd, J=8.03, 1.51 Hz, 1 H), 7.31 (t, J=7.91 Hz, 1 H), 7.24 (dd, J=7.65,1.63 Hz, 1 H), 3.74 (dt, J=13.87, 3.86 Hz, 2 H), 3.10-3.18 (m, 2 H),1.84-1.97 (m, 2 H), 1.72-1.84 (m, 2 H), 1.39 (s, 3 H). HRMS calcd forC₁₆H₁₉Cl₃N₅ (M+H)⁺ 386.0706, found 386.0691. IC₅₀ is 0.392 μM.

Example 246-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)-5-methylpyrazin-2-amine

A suspension of6-(4-amino-4-methylpiperidin-1-yl)-5-bromo-3-(2,3-dichlorophenyl)pyrazin-2-amine(20 mg, 0.046 mmol), potassium phosphate (29.5 mg, 0.139 mmol),PdCl₂(dppf).DCM adduct (1.9 mg, 0.002 mmol), and2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (7.8 μL, 0.056 mmol) inMeCN:H₂O (9:1, 500 μL, degassed) was stirred in a microwave reactor for1 h at 110° C. After cooling to RT, the volatiles were removed underreduced pressure and the resulting residue was purified by HPLC(gradient elution 25-50% acetonitrile in water, 5 mM NH₄OH modifier) togive the title compound (7.7 mg, 0.021 mmol). ¹H NMR (400 MHz,METHANOL-d₄) δ ppm 7.52 (dd, J=8.03, 1.76 Hz, 1 H), 7.31 (t, J=7.78 Hz,1 H), 7.23 (dd, J=7.65, 1.63 Hz, 1 H), 3.25-3.15 (m, 2 H), 3.01-3.13 (m,2 H), 2.25 (s, 3 H), 1.51-1.74 (m, 4 H), 1.14 (s, 3 H). HRMS calcd forC₁₇H₂₂Cl₂N₅ (M+H)⁺ 366.1252, found 366.1248. IC₅₀ is 0.222 μM.

Example 255-amino-3-(4-amino-4-methylpiperidin-1-yl)-6-(2,3-dichlorophenyl)pyrazine-2-carbonitrile

A solution of6-(4-amino-4-methylpiperidin-1-yl)-5-bromo-3-(2,3-dichlorophenyl)pyrazin-2-amine(50 mg, 0.116 mmol) and copper(I) cyanide (20.8 mg, 0.232 mmol) in DMF(500 μL) was stirred in a microwave reactor for 2 h at 180° C. Aftercooling to RT, the reaction mixture was filtered and the resultingsolution was purified by HPLC (gradient elution 25-50% acetonitrile inwater, 5 mM NH₄OH modifier) to give the title compound (22.0 mg, 0.053mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.64 (dd, J=7.96, 1.64 Hz, 1H), 7.42 (t, J=7.83 Hz, 1 H), 7.35 (dd, J=7.58, 1.77 Hz, 1 H), 3.83-4.04(m, 2 H), 3.62-3.83 (m, 2 H), 1.58-1.79 (m, 4 H), 1.25 (s, 3 H). HRMScalcd for C₁₇H₁₉Cl₂N₆ (M+H)⁺ 377.1048, found 377.1039. IC₅₀ is 0.315 μM.

Example 265-amino-3-(4-amino-4-methylpiperidin-1-yl)-6-(2,3-dichlorophenyl)pyrazine-2-carboxamide

A solution of5-amino-3-(4-amino-4-methylpiperidin-1-yl)-6-(2,3-dichlorophenyl)pyrazine-2-carbonitrile(38 mg, 0.101 mmol), NaOH (6 M in H₂O, 168 μL, 1.00 mmol), and hydrogenperoxide (103 μL, 1.00 mmol) in DMF (2 mL) was stirred in a microwavereactor for 1 h at 100° C. After cooling to RT, the reaction mixture wasfiltered and the resulting solution was purified by HPLC (gradientelution 15-40% acetonitrile in water, 0.1% TFA modifier) to give thetitle compound (12.8 mg, 0.034 mmol). ¹H NMR (400 MHz, DMSO-d₆) δ ppm7.65 (dd, J=7.53, 2.26 Hz, 1 H), 7.36-7.46 (m, 2 H), 7.32 (br. s., 1 H),6.91 (br. s., 1 H), 6.23 (s, 2 H), 3.37-3.52 (m, 4 H), 1.50-1.75 (br. s,2H), 1.36-1.53 (m, 4 H), 1.08 (s, 3 H). HRMS calcd for C₁₇H₂₁Cl₂N₆O(M+H)⁺ 395.1154, found 395.1140. IC₅₀ is 0.271 μM.

Example 273-(2,3-dichlorophenyl)-6-((3S,5R)-3,5-dimethylpiperazin-1-yl)pyrazin-2-amine

A suspension of3-bromo-6-((3S,5R)-3,5-dimethylpiperazin-1-yl)pyrazin-2-amine (55 mg,0.228 mmol), (2,3-dichlophenyl)boronic acid (52.1 mg, 0.273 mmol),potassium phosphate (145 mg, 0.683 mmol), and PdCl2(dppf).DCM adduct(18.6 mg, 0.023 mmol) in MeCN:H20 (9:1, 2.3 mL, degassed) was stirred ina microwave reactor for 1 h at 110° C. After cooling to RT, thevolatiles were removed under reduced pressure and the resulting residuewas purified by HPLC (gradient elution 25-50% acetonitrile in water, 5mM NH₄OH modifier) to give the title compound (17.0 mg, 0.048 mmol). ¹HNMR (400 MHz, METHANOL-d₄) δ ppm 7.74 (s, 1 H), 7.59 (dd, J=7.78, 1.51Hz, 1 H), 7.45 (dd, J=7.78, 1.51 Hz, 1 H), 7.38 (t, J=7.78 Hz, 1 H),3.56 (d, J=11.29 Hz, 2 H), 3.03-3.21 (m, 2 H), 2.44 (t, J=11.54 Hz, 2H), 1.17 (d, J=6.53 Hz, 6 H). HRMS calcd for C₁₆H₂₀Cl₂N₅ (M+H)⁺352.1096, found 352.1093. IC₅₀ is 0.570 μM.

The following compounds were made using the above procedure ormodifications to the above procedure using the correspondingintermediate and boronic acid. Although in most cases the Boc protectinggroup was removed under the reaction conditions, HCl or TFA was usedwhen necessary.

TABLE 4 Example Compound Characterization IC₅₀ (μM) 28

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.60 (d, J = 8.53 Hz, 2 H),7.25-7.32 (m, 2 H), 7.10-7.16 (m, 1 H), 6.50-6.55 (m, 1 H), 3.77-3.87(m, 2 H), 3.52-3.63 (m, 2 H), 1.60-1.75 (m, 4 H), 1.28 (s, 3 H). HRMScalcd for C₁₈H₂₂N₆ (M + H)⁺ 323.1906, found 323.1977. 0.632 29

as TFA salt. ¹H NMR (400 MHz, DMSO- d₆) δ ppm 7.39 (s, 1 H) 5.91 (dt, J= 3.73, 2.05 Hz, 1 H) 5.55 (s, 2 H) 3.49-3.61 (m, 2 H) 3.42 (ddd, J =13.01, 8.59, 4.17 Hz, 2 H) 2.22-2.31 (m, 2 H) 2.09-2.18 (m, 2 H)1.52-1.72 (m, 4 H) 1.26-1.47 (m, 4 H) 1.06 (s, 3 H). HRMS calcd forC₁₆H₂₆N₅ (M + H)⁺ 288.2188, found 288.2184. 2.585

Example 303-(4-amino-4-methylpiperidin-1-yl)-6-(2,3-dichlorophenyl)-1,2,4-triazin-5-amine

Step a: A suspension of(1-(5-amino-6-bromo-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate(250 mg, 0.646 mmol), 2,3-dichlorophenylboronic acid (154 mg, 0.807mmol), potassium phosphate (411 mg, 1.937 mmol), and XPhos 2^(nd)generation precatalyst (25.4 mg, 0.032 mmol) in MeCN:H₂O (9:1, 5 mL,degassed) was stirred in a microwave reactor for 2 h at 120° C. Aftercooling to RT, the reaction was filtered through a pad of Celitefollowed by EtOAc (20 mL) wash. The combined filtrates were concentratedand the resulting residue was purified by silica chromatography (0 to 5%gradient of MeOH(containing 1% NH₃)/DCM) to give tert-butyl(1-(5-amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate(78% purity). This mixture was further purified by HPLC (gradientelution 35-60% acetonitrile in water, 5 mM NH₄OH modifier) to givetert-butyl(1-(5-amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate(155 mg, 0.646 mmol). MS m/z 453.0 (M+H)⁺.

Step b: To a solution of tert-butyl(1-(5-amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate(155 mg, 0.342 mmol) in dioxane (2 mL), was added HCl (4 M in dioxane, 4mL). After stirring at RT until no starting material remained (monitoredby LCMS, the volatiles were removed under reduced pressure and theresulting residue was purified by silica chromatography (0 to 5%gradient of MeOH(containing 1% NH₃)/DCM) to give the title (130 mg,0.342 mmol, HCl salt). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.80 (dd,J=7.78, 2.01 Hz, 1 H), 7.42-7.58 (m, 2 H), 4.29 (m., 2 H), 3.64 (ddd,J=14.24, 9.47, 4.64 Hz, 2 H), 1.92-2.06 (m, 4 H), 1.57 (s, 3 H). HRMScalcd for C₁₅H₁₉Cl₂N₆ (M+H)⁺ 353.1048, found 353.1042. IC₅₀ is 0.194 μM.

Assays

Compounds of the invention were assessed for their ability toselectively inhibit SHP2 activity. The inhibitory properties of thecompounds of the invention described herein can be evidenced by testingin any one of the following assays.

SHP2 Allosteric Inhibition Assay

SHP2 is allosterically activated through binding ofbis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains.The latter activation step leads to the release of the auto-inhibitoryinterface of SHP2, which in turn renders the SHP2 protein tyrosinephosphatase (PTP) active and available for substrate recognition andreaction catalysis. The catalytic activity of SHP2 was monitored usingthe surrogate substrate DiFMUP in a prompt fluorescence assay format.

More specifically, the phosphatase reactions were performed at roomtemperature in 384-well black polystyrene plate, flat bottom, lowflange, non-binding surface (Corning, Cat #3575) using a final reactionvolume of 25 μL and the following assay buffer conditions: 60 mM HEPES,pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, 5 mM DTT.

The inhibition of SHP2 by compounds of the invention (concentrationsvarying from 0.003-100 μM) was monitored using an assay in which 0.5 nMof SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222(sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) (SEQ ID NO:1).After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP(Invitrogen, cat #D6567) was added to the reaction and incubated at 25°C. for 30 minutes. The reaction was then quenched by the addition of 5μl of a 160 μM solution of bpV(Phen) (Enzo Life Sciences cat#ALX-270-204). The fluorescence signal was monitored using a microplatereader (Envision, Perki-Elmer) using excitation and emission wavelengthsof 340 nm and 450 nm, respectively. The inhibitor dose response curveswere analyzed using normalized IC₅₀ regression curve fitting withcontrol based normalization. IC₅₀ results for compounds of the inventionare shown in examples and tables 1-7, above.

p-ERK Cellular Assay

p-ERK cellular assay using the AlphaScreen® SureFire™ Phospho-ERK 1/2Kit (PerkinElmer): KYSE-520 cells (30,000 cells/well) were grown in96-well plate culture overnight and treated with Shp2 inhibitors atconcentrations of 20, 6.6, 2.2, 0.74, 0.24,0.08, 0.027 μM for 2 hrs at37° C. Incubations were terminated by addition of 30 μL of lysis buffer(PerkinElmer) supplied with the SureFire phospho-extracellularsignal-regulated kinase (pERK) assay kit (PerkinElmer). Samples wereprocessed according to the manufacturer's directions. The fluorescencesignal from pERK was measured in duplicate using a 2101 multilabelreader (Perkin Elmer Envision). The percentage of inhibition wasnormalized by the total ERK signal and compared with the DMSO vehiclecontrol.

Colony Formation Assay and Cell Proliferation Assay

KYSE-520 Cells (1500 cells/well) were plated onto 24-well plates in 300μL medium (RPMI-1640 containing 10% FBS, Lonza). For drug treatment,compounds of the invention at various concentrations (20, 10, 5, 2.5,1.25 μM) were added 24 hours and 5 days after cell plating. At day 11,colonies were stained with 0.2% crystal violet (MP Biomedicals) andsubsequently dissolved in 20% acetic acid for quantitation using aSpectramax reader (Thermo Scientific). In cell proliferation assay,cells (1500-cells/well) were plated onto 96-well plates in 100 μL medium(RPMI-1640 containing 10% FBS, Lonza). At day 6, 50 μL Celltiter-Gloreagent (Promega) was added, and the luminescent signal was determinedaccording to the supplier's instruction (Promega).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

1. A method of treating a disease or disorder mediated by the activityof SHP2 comprising administering a compound of formula Ic:

in which: n is selected from 1, 2, 3, 4 and 5; Y₁ is selected from CHand N; Y₂ is CR₆; R_(3a) is selected from C₁₋₄alkyl, C₁₋₄alkoxy, amino,hydroxy, C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R_(4a) is selected fromhydrogen, C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy, C₃₋₈cycloalkyl andC₁₋₄alkyl-amino; R₆ is selected from hydrogen, halo and methyl; R₉ isselected from halo, amino, hydroxy, N₃, C₁₋₄alkyl,halo-substituted-C₁₋₄alkyl, C₁₋₄alkoxy, —C(O)OR₁₀ and —NHC(O)R₁₀; R₁₀ isselected from hydrogen, phenyl and naphthyl; wherein said phenyl of R₁₃is unsubstituted or substituted with methoxy; or a pharmaceuticallyacceptable salt thereof, to a person in need of such treatment in aneffective amount for the prophylactic or therapeutic treatment of thedisease or disorder.
 2. The method of claim 1, wherein the disease ordisorder mediated by the activity of SHP2 is selected from NoonanSyndrome, Leopard Syndrome, juvenile myelomonocytic leukemias,neuroblastoma, melanoma, acute myeloid leukemia, breast cancer,esophageal cancer, lung cancer, colon cancer, head cancer,neuroblastoma, squamous-cell carcinoma of the head and neck, gastriccarcinoma, anaplastic large-cell lymphoma and glioblastoma.
 3. Themethod of claim 2, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 4. A method of treatinglung cancer comprising administering the compound:

or a pharmaceutically acceptable salt thereof.